# More than a mutagenic Aflatoxin B1 precursor: The multiple cellular targets of Versicolorin A revealed by global gene expression analysis

Carine Al-Ayoubi, Ophélie Rocher, Claire Naylies, Yannick Lippi, Julien Vignard, Sylvie Puel, Olivier Puel, Isabelle P. Oswald, Laura Soler

<figure><img src="image_1.png" /></figure>

## To cite this version:

Carine Al-Ayoubi, Ophélie Rocher, Claire Naylies, Yannick Lippi, Julien Vignard, et al.. More than a mutagenic Aflatoxin B1 precursor: The multiple cellular targets of Versicolorin A revealed by global gene expression analysis. Environmental Pollution, 2024, 363, pp.125138. 10.1016/j.envpol.2024.125138. hal-04835843

HAL Id: hal-04835843

https://hal.inrae.fr/hal-04835843v1

Submitted on 19 Feb 2026

HAL is a multi-disciplinary open access archive for the deposit and dissemination of scientific research documents, whether they are published or not. The documents may come from teaching and research institutions in France or abroad, or from public or private research centers.

L'archive ouverte pluridisciplinaire HAL, est destinée au dépôt et à la diffusion de documents scientifiques de niveau recherche, publiés ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

<figure><img src="image_2.png" /></figure>
<figure><img src="image_3.png" /></figure>

<figure><img src="image_4.png" /></figure>

# More than a mutagenic Aflatoxin B1 precursor: The multiple cellular targets of Versicolorin A revealed by global gene expression analysis☆

Carine Al-Ayoubi, Ophelie Rocher, Claire Naylies, Yannick Lippi, Julien Vignard, Sylvie Puel, Olivier Puel, Isabelle P. Oswald, Laura Soler*

Toxalim (Research Centre in Food Toxicology), Université de Toulouse, INRAE, ENVT, INP, Purpan, UPS, 31027, Toulouse, France

## ARTICLE INFO

**Keywords:**

Mycotoxins
Versicolorin a
Aflatoxin B1
Transcriptome
Intestine

## ABSTRACT

Versicolorin A (VerA), a precursor of the potent carcinogen Aflatoxin B1 (AFB1), is an emerging mycotoxin. Recent research has highlighted the mutagenic and genotoxic properties of VerA, yet several facets of its pronounced toxicity remain unexplored. In the present study, we investigated early (6 h) transcriptomic changes induced by VerA in differentiated intestinal cells in non-cytotoxic conditions (1 and 3 µM) and compared its effects to those of AFB1 at 1 µM. Our findings indicated that VerA led to substantial alterations in global gene expression profiles, while AFB1 did not exhibit the same effects. As expected, both toxins caused alterations in gene expression associated with well-known aspects of their toxicity, including mutagenicity, genotoxicity, oxidative stress, and apoptosis. However, we also observed novel features of VerA toxicity, including the ability to cause mitochondrial dysfunction and to trigger a type-1 interferon response, at least partially mediated by cGAS-STING. VerA also induced changes in the expression of genes involved in the regulation of cell shape and adhesion, transcription/translation as well as genes associated with tumor biology. Our results provide new evidence of the high toxicity of VerA and underscore the importance of further assessing the risks associated with its presence in food.

## 1. Introduction

Aflatoxins, produced by toxigenic *Aspergillus* fungi, pose a significant global food safety threat (Bennett & Klich, 2003; Payros et al., 2021). These mycotoxins are widespread in hot and humid regions, contaminating various agricultural products such as cereals, nuts, seeds, and spices. Climate change projections indicate alarming increases in aflatoxin production in European maize crops (Bailly et al., 2018; Battilani et al., 2016). Among Aflatoxins, Aflatoxin B1 (AFB1) stands out as the most potent naturally occurring carcinogen (Schrenk et al., 2020). Its mode of action depends on its dihydrobisfuran structure, which is activated *in vivo* by cytochrome P450 enzymes and becomes a reactive mutagenic and genotoxic compound (Eaton et al., 2010; Smela et al., 2001). To address this threat, current global regulations concerning the presence of AFB1 in human food and animal feed authorize maximum levels of AFB1 ranging from 0.1 to 12 µg/kg depending on the product and its destination (European Communities, 2023).

The biosynthesis pathway of AFB1 is well known and involves the production of different bisfuranoid precursors that can accumulate in contaminated foodstuffs, including Versicolorin A (VerA) (Caceres et al., 2020; Jiang et al., 2019). Like AFB1, VerA is synthesized by aflatoxigenic *Aspergillus* species. Additionally, non-aflatoxigenic *Aspergillus* species produce VerA, explaining its detection at higher concentrations than AFB1 in certain food products (Abdallah et al., 2017; Gauthier et al., 2020; Janić Hajnal et al., 2020). VerA is an emerging mycotoxin and some of its toxicological characteristics have already been elucidated, but VerA is not yet subject to regulation. Like AFB1, VerA is highly mutagenic and genotoxic. Positive mutagenic responses have been demonstrated in bacterial mutation assays, when the effects were attributed to its metabolic activation in a potentially DNA-reactive form similar to that produced by AFB1 (Al-Ayoubi et al., 2022, 2023). Using metabolically competent and non-carcinogenic small intestinal cells (IPEC1) (Al-Ayoubi et al., 2023), we recently showed that a short (6 h) exposure to concentrations of VerA as low as 0.03 µM caused

<footnote>* This paper has been recommended for acceptance by Wen Chen.</footnote>

<footnote>* Corresponding author. UMR 1331 TOXALIM, INRA/INP/UPS Research Centre in Food Toxicology 180 chemin de Tournefeuille, BP.93173 F-31027, TOULOUSE, cedex 3, France.</footnote>

<footnote>E-mail address: laura.soler-vasco@inrae.fr (L. Soler).</footnote>
chromosomal damage and various pre-mutagenic lesions. Additional studies highlighted the greater complexity of VerA toxicity compared to that of AFB1, driven by certain key factors. For example, exposure to VerA induces higher levels of oxidative and replication stress in exposed cells than AFB1, which emerge as potentially important contributors to its genotoxicity, and are linked to the particular ability of VerA to induce DNA damage even in cells with poor metabolic activity (Gauthier et al., 2020; Theumer et al., 2018). In acellular models, VerA was also identified as a potent inducer of mitochondrial respiration uncoupling, warranting additional research (Kawai et al., 1983).

The present study aimed to understand the toxicity mechanisms of VerA in the intestine in order to improve our understanding of VerA toxicity, since present data suggest that VerA may have a more potent toxic effect than AFB1 and that the mechanisms underlying the toxicity of the two toxins are not the same.

## 2. Material and methods

### 2.1. Toxins

VerA was produced using an in-house method detailed in (Theumer et al., 2018). VerA was isolated from an *Aspergillus parasiticus* strain (SRRC 0164) cultivated on wheat grain, followed by purification using Ultimate 3000 high-performance liquid chromatography (HPLC; Thermo Fisher Scientific, Courtaboeuf, France). The identity and purity of the VerA toxin were determined by HPLC with a diode array detector and a Zorbax C18 analytical column, with VerA peaks representing 97% of the signals (Theumer et al., 2018). AFB1 was purchased from Sigma. Both toxins were dissolved in DMSO (Sigma) at 10 mM and stored at -20 °C until further use.

### 2.2. Cell culture and exposure conditions

The non-tumorigenic and non-transformed intestinal cell line IPEC1 was chosen as a model due to its competence in terms of cytochrome P450 and p53 status which made these cells suitable for the study of aflatoxins and other pro-toxicants (Nossol et al., 2015). The IPEC1 cells were grown as described elsewhere (Al-Ayoubi et al., 2023). IPEC1 cells were differentiated into monolayers using Transwell devices prior as previously described (Al-Ayoubi et al., 2022) and exposed apically to 1 µM and 3 µM VerA, 1 µM AFB1, or to control solvent (DMSO) for 6 h. The chosen concentrations of VerA represent the highest non-cytotoxic doses after 6 h of exposure (Al-Ayoubi et al., 2023), while AFB1 was used as a non-cytotoxic reference concentration, enabling comparison between the effects of VerA and AFB1 on transcriptome changes (Gauthier et al., 2020).

### 2.3. Microarray and functional analysis of differentially expressed genes

RNA isolation was performed as previously described (Pinton et al., 2010). Following extraction, the concentration and quality of the RNA were assessed using a Dropsense 96 UV/VIS droplet reader (Trinean, Belgium) and an Agilent 2100 Bioanalyzer (Agilent Technologies Inc., Santa Clara, CA, USA). The mean (±SD) RNA Integrity Number (RIN) was 9.53 ± 0.36. Gene expression profiling of the 16 samples (4 replicates per treatment group) were conducted at the GeT-TRiX platform (GénoToul, Génopole Toulouse Midi-Pyrénées) using Agilent Sureprint G3 Porcinet 60K_DEC2011 microarrays (8 × 60K, design 037880) in accordance with the manufacturer's instructions, as described in (Pierron et al., 2022). Microarray data and experimental details are available in NCBI's Gene Expression Omnibus (Edgar et al., 2002) and are accessible through GEO Series accession number GSE267572 (https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE267572).

## 2.4. Hypothesis validation: additional experiments

Several hypotheses on the intestinal toxicity of VerA were put forward in this study, deriving from transcriptomic assays and functional analyses. In order to validate these hypotheses, we have conducted several experiments detailed in Supplementary Data (Supplementary Methods).

## 2.5. Statistical analysis

Microarray data were analyzed using R (R Core Team, 2018) and Bioconductor packages (Huber et al., 2015) as described in GEO accession GSE267572. Raw data (median signal intensity) were filtered, log2 transformed and normalized using the quantile method (Bolstad et al., 2003). A model was fitted using the limma lmFit function (Ritchie et al., 2015). Pair-wise comparisons between biological conditions were applied using specific contrasts. A correction for multiple testing was applied using the Benjamini-Hochberg procedure (BH, Benjamini & Hochberg, 1995) to control the false discovery rate (FDR). Probes with FDR <5% were considered to be differentially expressed between conditions. The results were explored using MATRIX APP (Lippi & Soubès, 2023, https://matrix.toulouse.inrae.fr/). Hierarchical clustering was applied to the samples and the differentially expressed probes using 1-Pearson correlation coefficient as distance and Ward's criterion for agglomeration and the results are illustrated as a heat map. Functional analysis of the differentially expressed genes lists was performed using the EnrichR web server (Chen et al., 2013; Kuleshov et al., 2016; Xie et al., 2021) and Ingenuity Pathway Analysis tool (QIAGEN, Valencia, CA, USA). Only pathways with a P value < 0.05 are shown here.

# 3. Results and discussion

## 3.1. VerA, but not AFB1, induces marked changes in the gene expression profiles of differentiated intestinal epithelial cells

Exposure of intestinal cells to these conditions resulted in significant changes (FDR <0.05; fold-change >1) in gene expression compared to vehicle (DMSO) controls. AFB1 induced changes in 79 genes, while 1 µM VerA and 3 µM VerA induced changes in 3261 and 5211 genes, respectively (Fig. 1, A). Microarray results were confirmed by RT-qPCR analysis using selected genes (FLVCR2, SRM, MAPK2K6, SERPINB2 and MX2), and gene expression results obtained using the two techniques were in agreement (Table S3). Our study found that AFB1 exposure had a low impact on the transcriptome of intestinal cells, in contrast to the high impact of VerA. This observation aligns with previous research conducted on proliferating epithelial intestinal Caco-2 cells (Gauthier et al., 2020). However, it's important to note that our study identified fewer genes affected by VerA and AFB1 exposure compared to the aforementioned study. This discrepancy may be attributed to the non-proliferative state of the monolayer cells, as previously described for another mycotoxin (Hinze & Boucrot, 2018; Luo et al., 2021).

Principal component analysis (PCA) revealed that the concentration of VerA was the main source of variance (Fig. 1, B). The Venn diagram in Fig. 1, A reveals significant similarity in VerA-treated samples. Specifically, 2788 genes were identified in samples treated with both 3 µM and 1 µM VerA, accounting for 85.5% of all genes regulated at a VerA concentration of 1 µM. Additionally, 65 genes were found in both AFB1- and VerA-treated samples (at concentrations of both 3 µM and 1 µM), representing 82.3% of all genes regulated at an AFB1 concentration of 1 µM.

Next, double hierarchical clustering was performed to identify specific and shared expression profiles for differentially expressed probes (FDR <5%) identified in response to treatments. The results are presented in the form of a heatmap (Fig. 1, C). Three distinct gene clusters are highlighted as alternative grey levels next to the dendrogram on the left based on the relationships between their expression (Fig. 1, D). Cluster 1 included 2695 genes that were down-regulated in all VerA-
<figure><img src="image_5.png" /><figcaption>**Fig. 1.** Gene expression profile of intestinal cells exposed to AFB1 (1 µM), VerA (1 µM), VerA (3 µM). (A) Venn diagram representing the differentially expressed genes (Adjusted p-value <0.05) in cells exposed to 1 µM AFB1 (in green), 1 µM VerA (in purple) and 3 µM VerA (in pink) compared to solvent control cells. (B) Principal component analysis of normalized expression signals on all samples: 1 µM AFB1 (green), 1 µM VerA (purple) and 3 µM VerA (pink) and solvent control DMSO (black). (C) Heat map representing differentially expressed genes exposed to AFB1 (1 µM), VerA (1 µM), VerA (3 µM) compared to control (DMSO, adjusted p-value <0.05). Individual gene expression is represented in the heatmap, and the hierarchical clustering was obtained from individual expression values using Pearson correlation coefficient as distance and Ward's criterion for agglomeration. Red and green indicate values above and below the mean averaged centred and scaled expression values (Z-score), respectively. Black indicates values close to the mean. According to the probe clustering (left panel), 3 gene clusters showed specific gene expression profiles. (D) Violin plots with strip charts representing expression profiles of the three clusters identified by the clustering of differentially expressed probes illustrated in the heat map in (C). The average expression signal (z-score) of the probes is plotted for each of the four conditions: 1 µM AFB1 (green), 1 µM VerA (purple) and 3 µM VerA (pink) and solvent control DMSO (orange) cells.</figcaption></figure>

treated cells compared to in controls, although AFB1 had relatively weaker effects and the responses to VerA were dose dependent. Functional analysis (Table S4) revealed that the main regulated pathways of this cluster (i.e. those showing the most significant enrichment) were related to cadherin-mediated cell adhesion and changes in shape and their related signaling, as well as RNA processing. The predicted upstream regulators of the genes in this cluster were mainly TP53 and TGFB1. The 'top tox' list (set of molecules that are known to be involved in a particular type of toxicity) was primarily related to cell death. Notably, the list of 'top analysis-ready molecules' (an output list showing the genes with the strongest regulation; Table S5) mainly included genes involved in regulating cell adhesion and shape-related functions (DKK1, SPRED1, TUBB6, FGD6), and p53-related signaling (PPP1R1B3, PRRG1, NAV1).

Cluster 2 consisted of 726 genes that were upregulated at a similar magnitude in all treated cells. The differentially expressed genes in this cluster were mainly related to interferon (IFN) signaling, methionine and L-cysteine degradation, mesenchymal cell differentiation mediated by RUNX1 and caspase activation (Table S6). The primarily predicted upstream regulators of these processes were identified as poly rI:rC RNA and RNASEH2A. In the context of toxicity, the 'top tox' molecular set was primarily associated with oxidative stress. The genes that showed the strongest regulation in cluster 2 (Table S7) included several involved in processes related to tumorigenesis (TGFA, RDH16, UTRN), transcription regulation (KLF3), DNA repair (AHDC1) and apoptosis

(CASP5).

Cluster 3 included 1997 genes upregulated in all treated samples compared to in the control group but the magnitude of change in gene expression was only high in cells treated with VerA, mostly at a concentration of 3 µM. Functional analysis based on the most significant p-values, revealed that the main enriched pathways in this dataset were the sirtuin signaling pathway, mitochondrial dysfunction, IFN signaling, and processes related to DNA damage and repair (Table S8). TP53 and HNF4A were identified as main predicted upstream regulator in many of these processes. Additionally, the enriched GO molecular functions mainly included oxidized DNA binding and glutathione transferase activity. The genes showing the strongest regulation in cluster 3 (Table S9) are mainly known for their critical functions in tumor biology (NR3C2, SNTB1, ABLIM1, PPP1R9A, MBP and GHR). The list was completed by chaperones and genes involved in immune signaling (CCDC30, FLVCR2 and SLC7A7).

## 3.2. Both AFB1 and VerA induce overexpression of DNA damage response-related genes: mutagenicity as a common mechanism of action

Almost all the genes significantly regulated by AFB1 were also regulated by VerA at both concentrations tested (65 genes; Fig. 1, A) although the fold-changes in gene expression were more pronounced at a concentration of 3 µM VerA than at 1 µM VerA and that of AFB1 for these genes in all functional categories. The main function of these genes
was shown to be associated with DNA repair, cell cycle control and apoptosis. This result reflects the common toxicity of AFB1 and VerA as genotoxins. In our previous works, we demonstrated that VerA is likely to be biotransformed into a mutagenic derivative, which is known to be the main mechanism of action of AFB1 (Al-Ayoubi et al., 2022). Indeed, the signaling pathway ‘diseases of mismatched repair’ that is known to be activated in the presence of mutagenic agents, is overrepresented in our dataset. Further, both toxins can cause oxidative DNA damage (Al-Ayoubi et al., 2023). Our gene expression results consequently reflect the fact that both toxins can activate a DNA damage response (DDR) triggered by their mutagenicity.

According to our data, the DDR induced by both toxins seems to be mediated by ataxia telangiectasia-mutated - breast cancer 1 (ATM-BRCA1) and the tumor suppressor protein p53 pathways, potentially involving upstream regulators with roles already described during the cell response to AFB1, such as p53, HNF-4α (Marrone et al., 2016) and RNase H2. In response to DNA damage, p53 binds DNA and mediates transcriptional activation of genes whose products trigger cell-cycle arrest, DNA repair and/or apoptosis (Riley et al., 2008). The essential role of this tumor suppressor protein in the alleviation of the toxicity of AFB1 is known, and a mutation in codon 249 of the *TP53* gene is tightly linked with the development of hepatocarcinoma in individuals exposed to AFB1 (Hussain et al., 2007). While previous studies suggested that p53 is not essential for cell cycle control and apoptosis induction in intestinal cells exposed to VerA (Gauthier et al., 2020), we observed that 53BP1 is an important mediator of VerA-induced DDR, as it is recruited in IPEC1 cells exposed to VerA (Al-Ayoubi et al., 2023). According to the literature, some post-translational modifications are important regulators of p53 activity (Meek & Anderson, 2009) for example, Ser15 phosphorylation and Lys382 acetylation. Previous studies showed that AFB1 induces significantly higher levels of p53-Ser15 phosphorylation than VerA in Caco-2 and HepG2 cells (Budin et al., 2021; Gauthier et al., 2020), whereas in our study, only VerA induced a significant increase of Ser15-phosphorylated p53 levels in IPEC1 cells (at the highest concentration), while AFB1 did not (Fig. 2). What is more, only VerA induced

changes in gene expression in many p53-responsive genes including PPP1R1B, PRRG1, NAV1, CD82, PIK3R3, PIK3C2B and SESN1, suggesting that VerA activates p53 responses in exposed IPEC1 cells. Indeed, our results agree with previous findings indicating that wild-type p53 dynamics varies substantially between cell lines (Stewart-Ornstein & Lahav, 2017).

In addition to phosphorylation, Lys382 acetylation is a p53 transactivation pathway that enhances the sequence-specific DNA-binding activity of p53 in response to DNA damage, and is controlled by several NAD⁺-dependent deacetylases, including sirtuin 1 (SIRT1). Interestingly, we observed significant overrepresentation of the sirtuin signaling pathway (Fig. S5) and, in general, of genes exhibiting deacetylase activity in VerA-exposed cells. We examined the cytoplasmic and nuclear sirtuin-dependent deacetylase activity in IPEC1 cells exposed to 3 µM VerA. We detected a significant increase only in nuclear sirtuin-dependent deacetylase activity (Supplementary methods 3, Fig. 3, A), suggesting that sirtuin activity plays a key role in regulating cellular responses to VerA-induced stress and toxicity. Protein abundance levels of the nuclear sirtuin SIRT1 (Fig. 3, B) were also significantly increased in the presence of VerA (at both 3 µM and 1 µM), as well as in the presence of AFB1 (1 µM). In parallel with these results, we observed a concomitant VerA-dependent regulation of the expression of downstream genes within the SIRT1 pathway, such as *PPARGC-1*, *PPARG* and *FXR* (*p*-value <0.05). These data suggest that sirtuins, particularly SIRT1, play a role in the toxicity of VerA. Since SIRT1 mediates the acetylation of p53 and this posttranslational modification is important for the activity of p53 (Ren et al., 2019), we investigated whether VerA induces changes in Lys382-acetylation of p53. However, our findings revealed a non-significant accumulation of acetylated p53 in exposed cells (Fig. 2). More research is thus needed to understand the role of SIRT1 in VerA toxicity, which may involve transcriptional regulation of p53/histone acetylation (Zhang & Kraus, 2010), or be associated with mitochondrial toxicity (discussed below) and apoptosis induction (Tang, 2016).

Our results confirm that DDR is initiated by both AFB1 and VerA,

<figure><img src="image_6.png" /><figcaption>**Fig. 2.** Immunoblotting analysis of phospho-p53 and acetyl-p53 in intestinal cells exposed to AFB1 (1 µM), VerA (1 µM), VerA (3 µM) or not (CTRL). (A) Representative blots of 3 paired samples of phospho-p53, acetyl-p53 and p53 (B) Relative quantification of normalized signal (arbitrary units). Values of phospho-p53 and acetyl-p53 are relative to those of p53. Values are means with standard errors of the mean of the control represented by vertical bars (n = 3). Asterisks indicate statistical differences (*p* < 0.05).</figcaption></figure>
<figure><img src="image_7.png" /><figcaption>**Fig. 3.** Effects of VerA on the sirtuin signaling pathway in intestinal cells. (A) Deacetylase activity of sirtuins in subcellular fractions of intestinal cells exposed to 3 µM VerA for 6 h. Values are means with standard errors of the mean represented by vertical bars (n = 3). Asterisks indicate a significant difference (**)p < .01). (B) Immunoblotting analysis of sirtuin 1 in intestinal cells exposed to AFB1 (1 µM), VerA (1 µM), VerA (3 µM) or not (CTRL). (B, Left) Representative blots of 3 paired samples of sirtuin 1 (B, Right) Relative quantification of normalized signal (arbitrary units). Values are means with standard errors of the mean of the control represented by vertical bars (n = 3). Asterisks indicate statistical differences (**p < 0.01).</figcaption></figure>

confirming a common mechanism of action in these structurally related compounds, based on their mutagenicity. While this requires further studies, our data also suggest that, contrary to previously published results, p53 may be also an important mediator of DDR for VerA, as it is for AFB1.

### 3.3. VerA: an inhibitor of transcription?

Previous results indicated that VerA genotoxicity involves more mechanisms than that of AFB1, mainly the induction of a higher level of replication stress (Al-Ayoubi et al., 2023; Gauthier et al., 2020). Replication stress may explain the ability of VerA to induce genotoxicity in cell lines that lack metabolic activity, in contrast to AFB1 (Theumer et al., 2018). The results of functional analysis of our data are in line with this hypothesis and provide additional clues to how VerA could induce replication stress. We found an enrichment of molecular functions related to the biology of DNA and RNA in VerA-regulated genes. The RNA metabolism pathway, along with molecular functions related to RNA and snoRNA binding were significantly overrepresented. When we extracted the list of genes associated with the GO “RNA binding” from our dataset (Table S10), we observed that the expression of these genes is only regulated by VerA. Most of these genes, which are essential for correct transcription and translation, were significantly down-regulated, while the expression of the topoisomerases 1 and 2B was significantly up-regulated. Previous results (Gauthier et al., 2020) indicated that VerA exposure leads to the significant accumulation of

small nucleolar H/ACA RNA (SNORA) and C/D box RNA (SNORD) genes. Taken together, these results suggest that VerA may disrupt DNA/RNA biology, which at least partially explains the known activation of an early ATR response, increased FANCD2 nuclear accumulation and the overall high replication stress induced by VerA (Al-Ayoubi et al., 2023; Gauthier et al., 2020). Additional studies are needed to understand the mechanism by which VerA interferes with transcription and/or translation.

### 3.4. VerA induces higher oxidative stress than AFB1 depending on changes in gene expression

Oxidative stress was one of the ‘top tox’ to emerge from our functional analysis of genes in cluster 2, while oxidized DNA binding was identified as an enriched molecular function of genes in cluster 3. Additionally, two overrepresented pathways in cluster 2 were associated with enhanced degradation of methionine and its intermediary metabolite, L-cysteine. Since methionine plays a significant role as a scavenger of reactive oxygen species (ROS) (Luo and Levine, 2009), our results suggest toxin-mediated oxidative stress and, as a consequence, enhancement of methionine degradation. Oxidative stress occurs in AFB1-exposed cells as a consequence of ROS accumulation during the biotransformation of AFB1 into its reactive epoxide form (Koziel et al., 2021). This leads to activation of the antioxidant Nrf2/ARE pathway and depletion of the methionine cell pool. It is known that dietary methionine supplementation mitigates the toxicity of AFB1 by
modulating an Nrf2-mediated antioxidant pathway (Elwan et al., 2021). To check whether the predicted increased degradation of methionine was associated with changes in the expression of effector genes in the Nrf2 response (Table S11), we monitored expression of key genes in our dataset. Our analysis revealed that exposure to VerA was significantly associated with upregulation of CAT, GPX8, NQO1 and GCLM and with downregulation of SOD2, GPX1, GPX2, GPX3, HMOX1, GCLC and TXN. In contrast, exposure to AFB1 only caused significant downregulation of the intestinal glutathione peroxidase GPX2. These results suggest the presence of oxidative stress and the activation of Nrf2 responses in VerA but not in AFB1-treated cells. Indeed, compared to controls, IPEC1 cells treated with VerA at 3 µM for 6 h showed a significant accumulation of mitochondrial superoxide and inhibition of SOD activity (Fig. 4, B). This is in line with the results of previous studies showing that VerA induces high oxidative stress in intestinal cells, characterized by a significant accumulation of ROS, including hydrogen peroxide and superoxide anion (Gauthier et al., 2020).

### 3.5. VerA, but not AFB1, induces mitochondrial toxicity and an IFN-I response mediated, at least partly, by cGAS-STING

**3.5.1. Exposure to VerA induces mitochondrial damage.** Our transcriptomic results point at a VerA-specific induction of mitochondrial damage, a mechanism of toxicity already described in the literature (Kawai et al., 1983). Mycotoxins with a hydroxyl group at the β-position of their anthraquinone nucleus, like VerA, are known to have a strong uncoupling activity in mitochondrial respiration (Kawai et al., 1983;

Muto et al., 1997). Indeed, functional analysis indicated an increase in oxidoreductase activity and signs of mitochondrial dysfunction, particularly among the genes in cluster 3. Only VerA, mainly at the highest concentration, but not AFB1, regulated these genes associated with mitochondrial dysfunction. We observed that, among the genes regulated across all mitochondrial respiratory complexes, those in complex V underwent a more pronounced change in their fold-change ratio (Fig. S1).

The mitotoxicity of VerA was confirmed in our cell model. A significant drop in ATP levels was observed when IPEC1 cells were exposed to 0.6 and 1 µM VerA for 6 h while forced to only use oxidative phosphorylation to produce ATP compared to cells where glycolysis was enabled, under non-cytotoxic conditions (Fig. 4, A). At these concentrations, we observed a significant activation of caspase 3/7, effectors of the intrinsic apoptotic pathway. The activation of caspase 3/7 indicates mitochondrial dysfunction, being induced via mitochondrial damage and the activation of pro-apoptotic factors, pointing to VerA-induced mitochondrial alteration resulting in apoptosis (Fig. 4, A). Exposure to VerA (6 h; 3 µM) was also associated with a significant accumulation of mitochondrial superoxide and mitochondrial membrane depolarization (Fig. 4, B). Moreover, VerA (but not AFB1) promoted changes in genes related with mitophagy such as SQSTM1 and HSPD1. Immunoblot analysis showed that HIF-1, a protein known to respond to mitochondrial stress (Huang et al., 2023), accumulated in cells exposed to VerA, while BNIP3L, a protein degraded together with mitochondria during mitophagy, was significantly depleted (Fig. S6). All these findings provide strong evidence that VerA has mitotoxic properties, calling for

<figure><img src="image_8.png" /><figcaption>**Fig. 4. Induction of mitochondrial toxicity and oxidative stress of VerA in IPEC1 intestinal cells.** A. Measurement of ATP levels in IPEC1 intestinal cells exposed to different doses of VerA or vehicle (DMSO; CTRL) for 6 h in the presence or absence of glucose, evaluation of cytotoxicity (cell membrane integrity) and caspase 3/7 activation. B. Measurement of mitochondrial superoxide, mitochondrial membrane potential and superoxide dismutase activity in IPEC1 intestinal cells exposed to 1 or 3 µM of VerA, 1 µM of AFB1 or vehicle (DMSO; CTRL) for 6 h. Positive controls for each test are included, namely Antimycin (20 and 50 µM), FCCP and Menadione 20 µM, respectively. Values are means with standard errors relative to the mean of the control (n = 3). Asterisks indicate statistical differences (*p < .05; **p < .01; ***p < .001; ****p < .0001).</figcaption></figure>
characterization of its impact on cell respiration, metabolism and mitochondrial biogenesis.

### 3.5.2. Exposure to VerA induces the activation of cGAS-STING and an IFN-I response.
Our transcriptomics results clearly show an induction of the IFN-I response in cells exposed to VerA (Fig. S4). Indeed, we verified by RT-qPCR the overexpression of IFN-β1, the IFN receptor IFNGR2, the transcriptional regulators of type I IFN-dependent immune responses, and the IFN-inducible genes IRF3, GBP2, MX2, OAS1 and OAS2. While we cannot rule out involvement of ATM-dependent NFκB activation in response to genomic DNA damage, the modulation of genes associated with activation of IFN signaling responses observed in our dataset (Table S12) may also be attributed to VerA-specific mitochondrial dysfunction. IFN-I can be activated by the accumulation of ROS during mitochondrial toxicity, phenomena present in VerA-exposed cells (Fig. 4). In addition, this response can be mediated via the cyclic GMP-AMP synthase-stimulator of IFN response cGAMP interactor 1 (cGAS-STING) pathway. In fact, mitochondrial damage can lead to the release of mitochondrial DNA (mtDNA) in the cytoplasm, which is sensed by cGAS-STING (West et al., 2015). Our data strongly suggest that cGAS-STING is activated upon exposure to VerA, as we observed by RT-qPCR significant upregulation of STING, as well as the effector genes of this pathway TBK1, PTEN, PIAS1 and STAT1/2 (Table S12). Along with these changes, immunoblot analysis of cells exposed to VerA showed a significant increase in the abundance ratio phospho-STAT1/STAT1, a hallmark of the activation of cGAS-STING (Fig. S6). In line with these findings, our study indicates that similar pro-inflammatory signaling pathways engaged by mtDNA include the NLRP3 inflammasome and NF-κB pathways following exposure to VerA. Signs of activation of both pathways by VerA were validated via RT-PCR and immunoblot, such as higher gene expression of CASP1 and IL1B (Table S12), as well as accumulation of IKBα in exposed cells (Fig. S6). Overall, VerA is toxic to mitochondria, inducing a cascade of pro-inflammatory signals and oxidative stress, which highlights the importance of understanding the relevance of these events, as mtDNA-induced responses are implicated in the promotion of autoimmune, metabolic and degenerative diseases.

### 3.6. Is VerA cancerogenic? Evidence from gene expression analysis

As discussed above, VerA is mutagenic and genotoxic, and shares some features of toxicity with AFB1. The exposure conditions used in our study induce chromosomal damage and various pre-mutagenic lesions on proliferating IPEC1 cells (Al-Ayoubi et al., 2023). Yet, while AFB1 is known to be a cancerogenic compound, this is not the case for VerA. The present dataset provides information concerning the phenotypic changes that IPEC1 differentiated cells undergo after exposure to VerA that suggest a carcinogenic potential.

A group of genes in cluster 1 is functionally involved in the remodeling of epithelial adherent junctions, integrin signaling, and actin cytoskeleton signaling (Figs. S2 and S3). These genes were significantly downregulated (adjusted *p* < .05) in cells exposed to VerA in a concentration-dependent manner, but not in cells exposed to AFB1 (Table S5). These changes in gene expression suggest that epithelial cells exposed to VerA undergo phenotypical changes related to loss of adhesion, alterations to cell shape and to cell junctions. Functional analysis predicted that the upstream regulators of these changes would transform growth factor-beta (TGF-β; Table S4) and tumor protein p53 (TP53; Table S4). TGF-β is a potent growth suppressor of epithelial cells and mediates cell differentiation of intestinal epithelial cells (Morikawa et al., 2016), and the mutation of TP53 has been associated with, among other things, increased adhesion ability (Lee et al., 2015). In line with these findings, many genes in this cluster were found to be involved in cell growth, death control and motility (Table S5). The 10 top most regulated genes in this cluster code for proteins present in the cytoskeleton (TUBB6, FDG6), nucleosome (PROB1) or chaperone protein complexes (SDF2L1) or are else involved in p53 signaling (PPP1R13B,

PRRG1, NAV1, EEPD1) or in the regulation of epithelial-mesenchymal transition (EMT) initiation (DKK1, SPRED1). In line with these results, genes related to RUNX1-mediated signaling (RUNX1 regulates TGF-β induced EMT (Lu et al., 2020)); were overrepresented in cluster 2, and many genes involved in EMT initiation and progression, such as TGFA, SNTB1, ABLIM and PPP1R9A, were among the most regulated genes in clusters 2 and 3. EMT is a process in which polarized epithelial cells lose their epithelial phenotype and adopt a mesenchymal cell phenotype, including, among other changes, increased resistance to apoptosis, which is often associated with malignant transformation of epithelial cells (Koen & Collier, 2009).

Despite all the evidence for the induction of an EMT in differentiated intestinal cells in our dataset, we were not able to confirm modulation of CTNNβ and E-cadherin at the protein level. Furthermore, the abundance of other proteins, including the actin-regulating protein cofilin and the mesenchymal phenotype marker vimentin, did not change (Fig. S6). Because the short exposure time (6 h) may be insufficient to induce transition to a mesenchymal phenotype, studies with adjusted exposure conditions (non-cytotoxic concentrations, longer exposure) are now needed to determine by immunofluorescent staining whether VerA reduces the presence of membrane E-cadherin, causes trans-nucleation of β-catenin or promotes changes in the actin cytoskeleton related to an EMT transition, plus to identify the role of TGF-β in this phenomenon.

Interestingly, several of the genes most regulated by VerA were shown to be involved in tumor biology. The list includes different tumor suppressing genes such as RDH16, strongly regulated in hepatocellular cancer (Zhu et al., 2020), UTRN, which is mutated in a number of tumors (Li et al., 2007) and NFIB, also known to have tumor suppressive functions in many malignancies. Other genes known to be involved in colorectal tumorigenesis such as NR3C2, which is reported to function as a tumor suppressor (Li & Xu, 2022), GHR, which is frequently expressed in human colorectal cancers (Yang et al., 2004) and ABLIM1, which promotes growth and metastasis of colorectal cancer (He et al., 2024), were all modulated. This list is completed by SNTB1, which plays a crucial role in the stemness maintenance of cancer cells (Liang et al., 2021) and PPP1R9A, an imprinted gene, whose imprinting loss is linked with the development of different epithelial types of cancer (Hsu et al., 2016).

Overall, our results support the hypothesis that exposure to VerA may be associated with the induction of cell malignancy in intestinal cells, highlighting the need for further investigation into its effects on cellular transformations and underscoring the importance of exploring the carcinogenic potential of VerA.

## 4. Conclusion

Our study reveals new insights into VerA toxicity, confirming its greater toxicity to intestinal cells compared to AFB1. While VerA-induced transcriptome changes mirror those associated with DNA damage response, oxidative stress, and apoptosis seen with AFB1, VerA affects a larger number of genes and exhibits unique toxic aspects. Our data suggest that VerA's genotoxicity may involve replication stress due to RNA/DNA biology disruption. Additionally, VerA shows mitotoxicity in intestinal cells, potentially linked to IFN-I immune responses and inflammatory cascades. Dysregulation of genes related to a carcinogenic cell phenotype was also observed. Our findings support the need for in-vivo research, which is currently hampered by the lack of a method for producing or purifying large amounts of this non-commercial toxin. While this happens, and to further explore the carcinogenic potential of VerA in the intestine, more research employing chronic exposure settings, more sophisticated models (such as organoids, tissue explants), and other phenotyping approaches will be helpful. Overall, these findings contribute valuable toxicity data for assessing VerA exposure risk.
Declaration of generative AI and AI-assisted technologies in the
writing process

Statement: During the preparation of this work the author(s) used
COPILOT and QUILLBOT in order to reduce the length of the text
without losing information. After using this tool/service, the author(s)
reviewed and edited the content as needed and take(s) full responsibility
for the content of the published article.

CRediT authorship contribution statement

Carine Al-Ayoubi: Writing – original draft, Methodology, Investi-
gation, Formal analysis. Ophelie Rocher: Methodology. Claire Nay-
lies: Methodology, Conceptualization. Yannick Lippi: Writing – review
& editing, Formal analysis, Data curation, Conceptualization. Julien
Vignard: Writing – review & editing, Supervision, Formal analysis.
Sylvie Puel: Methodology. Olivier Puel: Writing – review & editing,
Funding acquisition, Conceptualization. Isabelle P. Oswald: Writing –
review & editing, Project administration, Funding acquisition, Concep-
tualization. Laura Soler: Vasco, Writing – review & editing, Writing –
original draft, Investigation, Funding acquisition, Conceptualization.

Declaration of competing interest

The authors declare the following financial interests/personal re-
lationships which may be considered as potential competing interests:
Soler L reports financial support was provided by French National
Research Agency. If there are other authors, they declare that they have
no known competing financial interests or personal relationships that
could have appeared to influence the work reported in this paper.

Acknowledgments

This research was supported in part by the ANR grants "Versitox"
(ANR-18-CE21-0009), "Precursors" (ANR-23-CE34-017) and the INRAE-
SA (National Research Institute for Agriculture, Food and Environment-
Animal Health Department) project "VersiTrans". The authors would
like to thank Daphne Goodfellow for English editing, Drerry
Gauthier for his scientific advice and Mrs Elham Atallah for her technical
help.

**Appendix A. Supplementary data**

Supplementary data to this article can be found online at https://doi.
org/10.1016/j.envpol.2024.125138.

Data availability

Microarray data and experimental details are available in NCBI's
Gene Expression Omnibus (Edgar et al., 2002) and are accessible
through GEO Series accession number GSE267572

References

Abdallah, M.F., Girgin, G., Baydar, T., Krska, R., Sulyok, M., 2017. Occurrence of multiple mycotoxins and other fungal metabolites in animal feed and maize samples from Egypt using LC-MS/MS. J. Sci. Food Agric. 97 (13), 4419–4428. https://doi.org/10.1002/jsfa.8293.

Al-Ayoubi, C., Alonso-Jauregui, M., Azqueta, A., Vignard, J., Mirey, G., Rocher, O., Puel, O., Oswald, I.P., Vettorazzi, A., Soler, L., 2023. Mutagenicity and genotoxicity assessment of the emerging mycotoxin Versicolorin A, an Aflatoxin B1 precursor. Environ. Pollut. 335, 122276. https://doi.org/10.1016/j.envpol.2023.122276.

Al-Ayoubi, C., Oules, J., Person, E., Bruel, S., Bouville, A., Pinton, P., Oswald, I.P., Jamin, E.L., Puel, O., Soler, L., 2022. Metabolism of versicolorin A, a genotoxic precursor of aflatoxin B1: characterization of metabolites using in vitro production of standards. Food Chem. Toxicol. 167 (June), 113272. https://doi.org/10.1016/j.fct.2022.113272.

Bailly, S., Mahgubi, A., Carvajal-Campos, A., Lorber, S., Puel, O., Oswald, I., Bailly, J.-D., Orlando, B., 2018. Occurrence and identification of *Aspergillus* section *flavi* in the

context of the emergence of aflatoxins in French maize. Toxins 10 (12), 525. https://
doi.org/10.3390/toxins10120525.

Battilani, P., Toscano, P., Van Der Fels-Klerx, H.J., Moretti, A., Camardo Leggieri, M., Brera, C., Rortais, A., Goumperis, T., Robinson, T., 2016. Aflatoxin B 1 contamination in maize in Europe increases due to climate change. Sci. Rep. 6, 1–7. https://doi.org/10.1038/srep24328.

Benjamini, Y., Hochberg, Y., 1995. Controlling the false discovery rate: a practical and powerful approach to multiple testing. J. Roy. Stat. Soc. B 57 (1), 289–300. https://doi.org/10.1111/j.2517-6161.1995.tb02031.x.

Bennett, J.W., Klich, M., 2003. Mycotoxins. Clin. Microbiol. Rev. 16 (3), 497–516. https://doi.org/10.1128/CMR.16.3.497-516.2003.

Bolstad, B.M., Irizarry, R.A., Astrand, M., Speed, T.P., 2003. A comparison of normalization methods for high density oligonucleotide array data based on variance and bias. Bioinformatics 19 (2), 185–193. https://doi.org/10.1093/bioinformatics/19.2.185.

Budin, C., Man, H.Y., Al-Ayoubi, C., Puel, S., van Vugt-Lussenburg, B.M.A., Brouwer, A., Oswald, I.P., van der Burg, B., Soler, L., 2021. Versicolorin A enhances the genotoxicity of aflatoxin B1 in human liver cells by inducing the transactivation of the Ah-receptor. Food Chem. Toxicol. 153, 112258. https://doi.org/10.1016/j.fct.2021.112258.

Caceres, I., Al Khoury, A., El Khoury, R., Lorber, S., Oswald, P., El Khoury, A.I., Atoui, A., Puel, O., Bailly, J.-D., 2020. Aflatoxin biosynthesis and genetic regulation: a review. Toxins 12 (3), 150. https://doi.org/10.3390/toxins12030150.

Chen, E.Y., Tan, C.M., Kou, Y., Duan, Q., Wang, Z., Meirelles, G.V., Clark, N.R., Ma'ayan, A., 2013. Enrich: interactive and collaborative HTML5 gene list enrichment analysis tool. BMC Bioinf. 14, 128. https://doi.org/10.1186/1471-2105-14-128.

Eaton, D.L., Beima, K.M., Bammler, T.K., Riley, R.T., Voss, K.A., 2010. Hepatotoxic mycotoxins. Comprehensive Toxicology 9, 527–569.

Elwan, H., Xie, C., Miao, L.P., Dong, X., Zou, X., Mohany, M., Ahmed, M.M., Al-Rejaie, S. S., Elnesr, S.S., 2021. Methionine alleviates aflatoxin B1-induced broiler chicks embryotoxicity through inhibition of caspase-dependent apoptosis and enhancement of cellular antioxidant status. Poultry Sci. 100 (8), 101103. https://doi.org/10.1016/j.pss.2021.101103.

European Communities, 2023. Commission Regulation (EU) 2023/915 of 25 April 2023 on maximum levels for certain contaminants in food and repealing Regulation (EC) No 1881/2006 (Text with EEA relevance). Off. J. Eur. Union L119, 103–157 (Document L:2023:119:TOC).

Gauthier, T., Duarte-Hospital, C., Vignard, J., Boutet-Robinet, E., Sulyok, M., Snini, S.P., Alassane-Kpembi, I., Lippi, Y., Puel, S., Oswald, I.P., Puel, O., 2020. Versicolorin A, a precursor in aflatoxins biosynthesis, is a food contaminant toxic for human intestinal cells. Environ. Int. 137, 105568. https://doi.org/10.1016/j.envint.2020.105568.

He, Y., Shi, Q., Ling, Y., Guo, H., Fei, Y., Wu, R., Tang, C., Zhang, X., Yao, L., 2024. ABLIM1, a novel ubiquitin E3 ligase, promotes growth and metastasis of colorectal cancer through targeting IκBα ubiquitination and activating NF-κB signaling. Cell Death Differ. 31 (2), 203–216. https://doi.org/10.1038/s41418-024-01256-y.

Hinze, C., Boucrot, E., 2018. Endocytosis in proliferating, quiescent and terminally differentiated cells. J. Cell Sci. 131 (23), 1–10. https://doi.org/10.1242/jcs.216804.

Hsu, C.M., Lin, P.M., Lin, H.C., Lai, C.C., Yang, C.H., Lin, S.F., Yang, M.Y., 2016. Altered expression of imprinted genes in squamous cell carcinoma of the head and neck. Anticancer Res. 36 (5), 2251–2258.

Huang, X., Zhao, L., Peng, R., 2023. Hypoxia-inducible factor 1 and mitochondria: an intimate connection. Biomolecules 13 (1), 1–14. https://doi.org/10.3390/biom13010050.

Hussain, S.P., Schwank, J., Staib, F., Wang, X.W., Harris, C.C., 2007. TP53 mutations and hepatocellular carcinoma: insights into the etiology and pathogenesis of liver cancer. Oncogene 26 (15), 2166–2176. https://doi.org/10.1038/sj.onc.1210279.

Janić Hajnal, E., Kos, J., Malachová, A., Steiner, D., Transka, M., Krská, R., Sulyok, M., 2020. Mycotoxins in maize harvested in Serbia in the period 2012–2015. Part 2: non-regulated mycotoxins and other fungal metabolites. Food Chem. 317. https://doi.org/10.1016/j.foodchem.2020.126409.

Jiang, M.P., Zheng, S.Y., Wang, H., Zhang, S.Y., Yao, D.S., Xie, C.F., Liu, D.L., 2019. Predictive model of aflatoxin contamination risk associated with granary-stored corn with versicolorin A monitoring and logistic regression. Food Addit. Contam. Part A Chemistry, Analysis, Control, Exposure and Risk Assessment 36 (2), 308–319. https://doi.org/10.1080/19440049.2018.1562226.

Kawai, K., Nozawa, Y., Maebayashi, Y., Yamazari, M., Hamasaki, T., 1983. The inhibition of mitochondrial respiration by anthraquinone mycotoxins. Averufin and Versicolorins A B. 1983 (18), 35–37. https://doi.org/10.2520/myco1975.1983.18_35.

Koen, E.J., Collier, A.B., 2009. The basics of epithelial-mesenchymal transition. Phys. Plasmas 119, 1420–1428. https://doi.org/10.1172/JCI39104.1420.

Koziel, M.J., Kowalska, K., Piastowska-Ciesielska, A.W., 2021. Nrf2: a main responsive element in cells to mycotoxin-induced toxicity. Arch. Toxicol. 95 (5), 1521–1533. https://doi.org/10.1007/s00204-021-02995-4.

Kuleshov, M.V., Jones, M.R., Rouillard, A.D., Fernandez, N.F., Duan, Q., Wang, Z., Koplev, S., Jenkins, S.L., Jagodnik, K.M., Lachmann, A., McDermott, M.G., Monteiro, C.D., Gundersen, G.W., Maayan, A., 2016. Enrich: a comprehensive gene set enrichment analysis web server 2016 update. Nucleic Acids Res. 44 (1), 90–97. https://doi.org/10.1093/nar/gkw377.

Lee, J.G., Ahn, J.H., Kim, T.J., Lee, J.H., Choi, J.H., 2015. Mutant p53 promotes ovarian cancer cell adhesion to mesothelial cells via integrin β4 and Akt signals. Sci. Rep. 5, 1–12. https://doi.org/10.1038/srep12642.

Li, J., Xu, Z., 2022. NR3C2 suppresses the proliferation, migration, invasion and angiogenesis of colon cancer cells by inhibiting the AKT/ERK signaling pathway. Mol. Med. Rep. 25 (4), 1–8. https://doi.org/10.3892/mmr.2022.12649.
Li, Y., Huang, J., Zhao, Y.L., He, J., Wang, W., Davies, K.E., Nosé, V., Xiao, S., 2007. UTRN on chromosome 6q24 is mutated in multiple tumors. Oncogene 26 (42), 6220–6228. https://doi.org/10.1038/sj.onc.1210432.

Liang, Y., Wang, B., Chen, S., Ye, Z., Chai, X., Li, R., Li, X., Kong, G., Li, Y., Zhang, X., Che, Z., Xie, Q., Lian, J., Lin, B., Zhang, X., Huang, X., Huang, W., Qiu, X., Zeng, J., 2021. Beta-1 syntrophin (SNTB1) regulates colorectal cancer progression and stemness via regulation of the Wnt/β-catenin signaling pathway. Ann. Transl. Med. 9 (12), 1016. https://doi.org/10.21037/atm-21-2700.

Lippi, Y., Soubès, F., 2023. MATRIX: a Shiny Application for Mining and Functional Analysis of Transcriptomics Data swh:1:dir: a78cb8f1c0fca114d6fc7fcdcf6659545570da1b). (hal-04251787).

Lu, C., Yang, Z., Yu, D., Lin, J., Cai, W., 2020. RUNX1 regulates TGF-β induced migration and EMT in colorectal cancer. Pathol. Res. Pract. 216 (11), 153142. https://doi.org/10.1016/j.prp.2020.153142.

Luo, Shen, Levine, R.L., 2009. Methionine in proteins defends against oxidative stress. Faseb. J. 23 (2), 464–472. https://doi.org/10.1096/fj.08-118414.

Luo, S., Terciolo, C., Neves, M., Puel, S., Naylies, C., Lippi, Y., Pinton, P., Oswald, I.P., 2021. Comparative sensitivity of proliferative and differentiated intestinal epithelial cells to the food contaminant, deoxynivalenol. Environ. Pollut. 277, 116818. https://doi.org/10.1016/j.envpol.2021.116818.

Marrone, A.K., Tryndyak, V., Beland, F.A., Pogribny, I.P., 2016. MicroRNA responses to the genotoxic carcinogens aflatoxin B1 and benzo[a]pyrene in human HepaRG cells. Toxicol. Sci. 149 (2), 496–502. https://doi.org/10.1093/toxsci/kfv253.

Meek, D.W., Anderson, C.W., 2009. Posttranslational modification of p53: cooperative integrators of function. Cold Spring Harbor Perspect. Biol. 1 (6), 1–16. https://doi.org/10.1101/cshperspect.a000950.

Morikawa, M., Derynck, R., Miyazono, K., 2016. TGF-B and the TGF-b family: context-dependent roles in cell and tissue physiology. Cold Spring Harbor Perspect. Biol. 8, 1–24. https://doi.org/10.1101/cshperspect.a021873.

Muto, Y., Matsunami, M., Kawai, K., Hamasaki, T., 1997. The effect of versicolorin A on electrical conductance in planar lipid bilayer membranes. Mycotoxins 1997 (44), 45–48. https://doi.org/10.2520/myco1975.199745.

Nossol, C., Barta-böszörményi, A., Kahlert, S., Zuschratter, W., 2015. Comparing two intestinal porcine epithelial cell lines (IPECs): morphological differentiation, function and metabolism. PLoS One 10 (7), 1–20. https://doi.org/10.1371/journal.pone.0132323.

Payros, D., Garofalo, M., Pierron, A., Soler-Vasco, L., Al-Ayoubi, C., Maruo, V.M., Alassane-Kpembi, I., Pinton, P., Oswald, I.P., 2021. Mycotoxins in human food: a challenge for research. Cah. Nutr. Diet. 56, 170–183. https://doi.org/10.1016/j.cnd.2021.02.001.

Pierron, A., Neves, M., Puel, S., Lippi, Y., Soler, L., Miller, J.D., Oswald, I.P., 2022. Intestinal toxicity of the new type A trichothecenes, NX and 3ANX. Chemosphere 288 (P1), 132415. https://doi.org/10.1016/j.chemosphere.2021.132415.

Pinton, P., Braicu, C., Nougayrede, J.P., Laffitte, J., Taranu, I., Oswald, I.P., 2010. Deoxynivalenol impairs porcine intestinal barrier function and decreases the protein expression of claudin-4 through a mitogen-activated protein kinase-dependent mechanism. J. Nutr. 140 (11), 1956–1962. https://doi.org/10.3945/jn.110.123919.

R Core Team, 2018. A Language and Environment for Statistical Computing. R Foundation for Statistical Computing, Vienna.

Ren, Z., He, H., Zuo, Z., Xu, Z., Wei, Z., Deng, J., 2019. The role of different SIRT1-mediated signaling pathways in toxic injury. Cell. Mol. Biol. Lett. 24 (1), 1–10. https://doi.org/10.1186/s11658-019-0158-9.

Riley, T., Sontag, E., Chen, P., Levine, A., 2008. Transcriptional control of human p53-regulated genes. Nat. Rev. Mol. Cell Biol. 9 (5), 402–412. https://doi.org/10.1038/nrm2395.

Ritchie, M.E., Phipson, B., Wu, D., Hu, Y., Law, C.W., Shi, W., Smyth, G.K., 2015. Limma powers differential expression analyses for RNA-sequencing and microarray studies. Nucleic Acids Res. 43 (7), e47. https://doi.org/10.1093/nar/gkv007.

Schrenk, D., Bignami, M., Bodin, L., Chipman, J.K., del Mazo, J., Grasl-Kraupp, B., Hogstrand, C., Hoogenboom, L., Leblanc, J.C., Nebbia, C.S., Nielsen, E., Ntzani, E., Petersen, A., Sand, S., Schwerdtle, T., Vlemminckx, C., Marko, D., Oswald, I.P., Piersma, A., et al., 2020. Scientific opinion – risk assessment of aflatoxins in food. EFSA J. 18 (3), 112. https://doi.org/10.2903/j.efsa.2020.6040.

Smela, M.E., Currier, S.S., Bailey, E.A., Essigmann, J.M., 2001. The chemistry and biology of aflatoxin B1: from mutational spectrometry to carcinogenesis. Carcinogenesis 22 (4), 535–545. https://doi.org/10.1093/carcin/22.4.535.

Stewart-Ornstein, J., Lahav, G., 2017. P53 dynamics in response to DNA damage vary across cell lines and are shaped by efficiency of DNA repair and activity of the kinase ATM. Sci. Signal. 10 (476). https://doi.org/10.1126/scisignal.aah6671.

Tang, B.L., 2016. Sirt1 and the mitochondria. Mol. Cell. 39 (2), 87–95. https://doi.org/10.14348/molcells.2016.2318.

Theumer, M.G., Henneb, Y., Khoury, L., Snini, S.P., Tadrist, S., Canlet, C., Puel, O., Oswald, I.P., Audebert, M., 2018. Genotoxicity of aflatoxins and their precursors in human cells. Toxicol. Lett. 287, 100–107. https://doi.org/10.1016/j.toxlet.2018.02.007.

West, A.P., Khoury-hanold, W., Staron, M., Tal, M.C., Cristiana, M., Lang, S.M., Bestwick, M., Duguay, B.A., Raimundo, N., Macduff, D.A., Kaech, S.M., Smiley, J.R., Means, R.E., Shadel, G.S., 2015. Mitochondrial DNA stress primes the antiviral innate immune response. Nature 520 (7548), 553–557. https://doi.org/10.1038/nature14156.

Xie, Z., Bailey, A., Kuleshov, M.V., Clarke, D.J.B., Evangelista, J.E., Jenkins, S.L., Lachmann, A., Wojciechowicz, M.L., Kropiwnicki, E., Jagodnik, K.M., Jeon, M., Ma'ayan, A., 2021. Gene set knowledge discovery with enrichr. Current Protocols 1 (3), 1–84. https://doi.org/10.1002/cp21.90.

Yang, X., Liu, F., Xu, Z., Chen, C., Li, G., Wu, X., Li, J., 2004. Growth hormone receptor expression in human colorectal cancer. Dig. Dis. Sci. 49 (3), 1493–1498. https://doi.org/10.1023/B:DDAS.0000042254.35986.57.

Zhang, T., Kraus, W.L., 2010. SIRT1-dependent regulation of chromatin and transcription: linking NAD+ functions. Biochim. Biophys. Acta 1804 (8), 1666–1675. https://doi.org/10.1016/j.bbpap.2009.10.022.

Zhu, Y.-H., Li, J.-B., Wu, R.-Y., Yu, Y., Li, X., Li, Z.-L., Zhang, H.-L., Feng, G.-K., Deng, R., Zhu, X.-F., 2020. Clinical significance and function of RDH16 as a tumor-suppressing gene in hepatocellular carcinoma. Hepatology Research 50, 110–120. https://doi.org/10.1111/hepr.13432.