Insights into cellular metabolic pathways of the combined toxicity responses of Caco-2 cells exposed to deoxynivalenol, zearalenone and Aflatoxin B₁

Highlights

• Metabolic profiling in Caco-2 cells was studied for the combined toxic effects of mycotoxins.

• The combined DON + AFB1 and DON + ZEN + AFB1 treatment has an obvious “synergistic effect”.

• Combined mycotoxins downregulate Bcl-2 gene and upregulate Bax, p53, caspase-3, caspase-8 and caspase- 9 genes.

• Combined mycotoxins seriously effect glycine, serine and threonine metabolism, pyruvate metabolism.

Abstract

Metabolic profiling in Caco-2 cells was studied for the combined toxic effects of deoxynivalenol (DON), zearalenone (ZEN), and Aflatoxin B₁ (AFB₁) through untargeted GC-MS. The GC-MS spectra of Caco-2 cells treated with individual 6.7 μM DON, 20 μM ZEN, 20 μM AFB₁ and the combined DON + AFB₁ (6.7 + 20 μM) and DON + ZEN + AFB₁ (6.7 + 20 + 20 μM) for 24 h were deconvoluted, aligned and identified with MS DIAL. The metabolic pathway analysis was analyzed with MetaMapp and visualized with CytoScape. Results show that the combined DON + AFB₁ and DON + ZEN + AFB₁ treatment has an obvious “synergistic effect”. The apoptosis-related gene mRNA test result indicates that the combined mycotoxins downregulate Bcl-2 gene and upregulate Bax, p53, caspase-3, caspase-8 and caspase-utilized 9 genes, more significantly than any individual mycotoxins group. The cellular metabolism illustrated that the combined mycotoxin groups, DON + ZEN + AFB₁ seriously effect glycine, serine and threonine metabolism, pyruvate metabolism, etc. while no metabolic disorders were presented in individual mycotoxin group. Our hypothesis was validated that the combined mycotoxins with low concentrations can have a synergistic effect in the metabolism, which may lead to cellular apoptosis or necrosis.

Introduction

Mycotoxins are ubiquitously present in agricultural commodities. Although there are almost four hundred mycotoxins known today, only a few mycotoxin groups have received widespread attention (Berthiller et al., 2013). The most important mycotoxins or mycotoxin groups are aflatoxins (AFs), zearalenone (ZEN), deoxynivalenol (DON), fumonisins (FUM) and ochratoxin A (OTA) (Streit et al., 2013). Among these, AFB₁ is the most toxic and carcinogenic, which could cause various diseases, including liver cancer, effects on the reproductive system and the immune system, encephalopathy with fatty degeneration of viscera, and pulmonary interstitial fibrosis (Marin et al., 2013; Lei et al., 2013). DON belongs to the type-B trichothecene group. Although it is not as toxic as other trichothecenes, it is one of the most common contaminants of cereals worldwide. DON directly binds to ribosome and causes translation, which activates stress kinases by ribotoxic stress response (Wen et al., 2016). Meanwhile, acute effects in humans are abdominal pains, headache, dizziness, throat irritation, nausea and many others (Rotter, 1996). ZEN competitively associates with estrogen receptors causing reproductive disorders, genotoxicity and testicular toxicity in some animal species and possibly human as well. Among the grains for human consumption, ZEN often occur in maize (Zinedine et al., 2007).

The mycotoxin survey (2005–2012) consisting of 19,000 samples collected from all over the world have been analyzed showing that 72% of the samples contained detectable amounts of mycotoxins and 38% of all samples contained two or more mycotoxins (Schatzmayr and Streit, 2013). A full 94% of samples contained at least one mycotoxin, and 76% of samples contained two or more mycotoxins which could impact human and animal health at already low doses (Muccio, 2017). Co-occurrence of mycotoxins is likely to arise because (i) most fungi can simultaneously produce multiple mycotoxins, (ii) commodities can be contaminated by several fungi at the same time, and (iii) completed feed is made from various commodities (Jelinek et al., 1989; Streit et al., 2012). The data on combined toxic effects of mycotoxins are limited and the health risk from multi-exposure is not well-known. Therefore, an increasing number of mycotoxin studies are devoted to their combined toxicity, especially to the exploration of the type of toxicological interaction (Lee and Ryu, 2017). DON, ZEN, and AFB₁ are so ubiquitous and co-occur in a range of commodities. However, there is few papers that study their combined cytotoxic properties. It is therefore necessary to assess the combined toxicity of these three mycotoxins. These cells are the first to be exposed to mycotoxins and at higher doses than other tissue cells. Cell submicroscopic studies have shown that Caco-2 cells are morphologically similar to human intestinal epithelial cells with identical cell polarity and tight junctions. And detection of pinocytosis function also showed that Caco-2 cells are similar to human intestinal epithelial cells. To study the effect of mycotoxin mixtures on the gastrointestinal tract, we selected the human intestinal cells Caco-2 as the in vitro experimental system.

Metabolomics is analytical technique that investigates small molecules present within a biological sample isolated from animal or cultured cells (Fiehn, 2002). Metabolomics is a well-established methodology which based on GC-MS and/or HPLC-MS techniques assisted with variety of statistical analysis, which can be an extremely powerful when elucidating the specific metabolic changes within a biological system in response to environmental changes such as disease, infection, or toxins (Hayton et al., 2017; Castro-Puyana and Herrero, 2013). Gas chromatography−mass spectrometry (GC-MS)−based metabolomics is ideal for identifying and quantitating small-molecule metabolites ( < 650 Da), including small acids, alcohols, hydroxyl acids, amino acids, sugars, fatty acids, sterols, catecholamines, drugs, and toxins, often using chemical derivatization to make these compounds sufficiently volatile for gas chromatography (Fiehn, 2016). Metabolomics of cultured cells has the potential to uncover previously unknown information about cell biology, functions and response mechanisms (Zhang et al., 2013). However, only few studies on animal or human biological system response to mycotoxin have been reported (De Pascali et al., 2017).

Understanding the potential effects of mixtures of mycotoxins is a complex task that requires a sound theoretical framework which can be tested experimentally. The CI models were used to predict the combined effects of DON, ZEN, and AFB₁. Metabolomics is utilized to evaluate and explain the biological systems changes. To the best of our knowledge, this work is the first study which utilizes metabolomics platform to uncover cellular mechanisms and cellular toxicity in response to individual and combined mycotoxins exposure.