Enniatin A

Exposure of humans and animals to mycotoxins via food and feed generally involves a conglomeration of compounds contaminating the consumed products. Investigations on combinatory effects of mycotoxins are therefore of great importance. In this study, cytotoxic effects of binary mixtures of the Fusarium toxins enniatin B, aurofusarin, deoxynivalenol, nivalenol and zearalenone, and tenuazonic acid produced by Alternaria spp., were evaluated by the WST-1 assay in the colorectal carcinoma cell-line Caco-2 after 24h of incubation. The selection of these mycotoxins was based on typically occurring natural contamination patterns in grains. Aurofusarin, which can be found abundantly in contaminated foodstuff and has not been toxicologically characterized properly so far, showed pronounced cytotoxicity, decreasing the mitochondrial activity at 10μM to 51% compared to a solvent control. Combinations of other mycotoxins with aurofusarin showed additive effects. In contrast, binary mixtures of enniatin B, deoxynivalenol, nivalenol and zearalenone at cytotoxic concentrations, predominantly resulted in antagonistic effects. Binary combinations of these four Fusarium toxins with tenuazonic acid also revealed interacting effects leading to a decrease in cytotoxicity, compared to expected combinatory effects. Especially in combination with deoxynivalenol, tenuazonic acid was found to significantly reduce the cytotoxicity of this mycotoxin in Caco-2 cells. Synergistic effects were not observed for any toxin combination under the chosen conditions.

Enniatins (ENN) and beauvericin (BEA) exert cytotoxic properties. Here, we observed that their impact on Ca2+‐homeostasis can be reversed by exogenous ATP. Thus, we investigated whether membrane‐located ATP‐binding cassette (ABC) transporters influence ENNs‐ and BEA‐induced cytotoxicity. In short‐term exposure assays breast cancer resistance protein (ABCG2)‐overexpression weakly but significantly reduced the cytotoxic activity of BEA but not ENNs. In contrast, multidrug resistance‐associated protein‐1 (ABCC1)‐ and P‐glycoprotein (ABCB1)‐overexpression was not protective under identical conditions. ABCG2‐mediated resistance against BEA was reversible by ABCG2 modulators. In long‐term exposure assays, ABCG2 and ABCB1 significantly protected against ENNs‐ and to a lesser extent BEA‐induced cytotoxicity. Moreover, both fusariotoxins potently inhibited the ABCG2‐ and ABCB1‐mediated efflux of specific fluorescent substrates, with BEA being more effective. Additionally, ATPase and photoaffinity‐labelling assays proofed interaction of both substances with ABCG2 and ABCB1. Remarkably, 2 years selection of KB‐3‐1 cells against both fusariotoxins resulted only in two‐fold ENNs but negligible BEA resistance. Interestingly, the selected sublines displayed upregulation of multidrug resistance proteins and crossresistance to other chemotherapeutics. Summarizing, ABCG2 and ABCB1 slightly but significantly protect human cells against ENNs‐ and BEA‐induced cytotoxicity. However, both mycotoxins potently interact with ABCB1 and ABCG2 transport functions suggesting influences on bioavailability of xenobiotics and pharmaceuticals.