Ex Vivo and In Vitro Studies Revealed Underlying Mechanisms of Immature Intestinal Inflammatory Responses Caused by Aflatoxin M1 Together with Ochratoxin A.

Zi-Wei Wang,Jia-Qi Wang,Sheng-Nan Huang,Nan Zheng,Ya-Nan Gao

doi:10.3390/toxins14030173

Abstract

Aflatoxin M1 (AFM1) and ochratoxin A (OTA), which are occasionally detected in milk and commercial baby foods, could easily enter and reach the gastrointestinal tract, posing impairment to the first line of defense and causing dysfunction of the tissue. The objective of this study was to investigate the immunostimulatory roles of individual and combined AFM1 and OTA on the immature intestine. Thus, we used ELISA assays to evaluate the generation of cytokines from ex vivo CD-1 fetal mouse jejunum induced by AFM1 and OTA and explored the related regulatory pathways and pivot genes using RNA-seq analysis. It was found that OTA exhibited much stronger ability in stimulating pro-inflammatory cytokine IL-6 from jejunum tissues than AFM1 (OTA of 4 μM versus AFM1 of 50 μM), whereas the combination of the two toxins seemed to exert antagonistic actions. In addition, transcriptomics also showed that most gene members in the enriched pathway ‘cytokine–cytokine receptor interaction’ were more highly expressed in OTA than the AFM1 group. By means of PPI network analysis, NFKB1 and RelB were regarded as hub genes in response to OTA but not AFM1. In the human FHs 74 Int cell line, both AFM1 and OTA enhanced the content of reactive oxygen species, and the oxidative response was more apparent in OTA-treated cells in comparison with AFM1. Furthermore, OTA and AFM1 + OTA raised the protein abundance of p50/RelB, and triggered the translocation of the dimer from cytosol to nucleus. Therefore, the experimental data ex vivo and in vitro showed that OTA-induced inflammation was thought to be bound up with the up-regulation and translocation of NF-κB, though AFM1 seemed to have no obvious impact. Since it was the first attempt to uncover the appearances and inner mechanisms regarding inflammation provoked by AFM1 and OTA on immature intestinal models, further efforts are needed to understand the detailed metabolic steps of the toxin in cells and to clarify their causal relationship with the signals proposed from current research.

Highlights

Mycotoxins are secondary metabolites generated by several range of fungal genera spp., such as Aspergillus, Penicillium, Fusarium, Alternaria, and Claviceps, which could induce toxic and even carcinogenic outcomes in the human body [1]
Toxins 2022, 14, 173 the results of Enzyme-linked immunosorbent assay (ELISA) assays that Aflatoxin M1 (AFM1) higher than 50 μM and ochratoxin A (OTA) of 4 μM significantly induced the release of IL-6 from the jejunum (p < 0.05), while AFM1 and OTA at all dosages decreased the level of IL-10 (Figure 1D,E and Supplementary Material Figure S1A,B)
Compared to no treatment group, OTA higher than 4 μM significantly increased the concentrations of TNF-α, whereas AFM1 dose-independently increased TNF-α levels without remarkable differences (Figure 1G,H)

Summary

Introduction

Mycotoxins are secondary metabolites generated by several range of fungal genera spp., such as Aspergillus, Penicillium, Fusarium, Alternaria, and Claviceps, which could induce toxic and even carcinogenic outcomes in the human body [1]. Infants are more susceptible to the toxic effects of mycotoxins, for the sake of developing immune, digestive, and detoxification systems, fairly restricted diet, higher rate of metabolism, and lower body weight [2–4]. It has been reported that exposure to mycotoxins from breast milk could create a lower risk than direct intake through breast milk surrogates [5]. The introduction of complementary foods usually occurs earlier than the recommended age of six months in many low- and middle-income countries, which tends to increase mycotoxins exposure during infancy [6,7]. What’s more, early infant co-exposure of mycotoxins from the same or different food sources may occur, posing additional health risks to the vulnerable population group arising from the synergetic toxicity of some mycotoxins [8,9]

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