The Combined Effect of Two Alternaria Mycotoxins (Alternariol and Alternariol Monomethyl Ether) on Porcine Epithelial Intestinal Cells

Alternariol (AOH) and alternariol monomethyl ether (AME) are secondary metabolites produced by fungi belonging to the genus Alternaria, which generally contaminate fruits but also cereal crops and vegetables. The objective of this study was to investigate if the co-exposure of the swine epithelial intestinal cell line (IPEC-1) to a mixture of mycotoxins would cause an increase in toxicity as compared with exposure to a single toxin. The effects of individual toxins as well as those of their combination (1:1 ratio), in a range of 1–250 μM, were assessed in vitro for the cell viability of proliferating IPEC-1 cells and then on parameters related to the oxidative stress. Our results indicate that both AOH and AME significantly decreased the IPEC-1 cell viability, but the cytotoxicity induced by the AOH + AME combination was significantly higher than that induced by the exposure to the individual toxins. The main interaction type ranged from slight synergy for the AOH-AME combination affecting 25% of cell viability (CI = 0.88), which evolved into a synergistic effect for a higher level of cytotoxicity IL50 (CI = 0.41) and a strong synergistic effect at IL90 (CI = 0.10). In addition, we investigated the effects of two low concentrations (2.5 μM and 5 μM) of AOH and AME mycotoxins administered individually or in combination on oxidative stress in IPEC-1 cells. Both AOH and AME can induce an increase in reactive oxygen species—ROS (+) cells%—and oxidative damage in porcine IPEC-1 cells. At least an additive effect was observed when the cells were exposed to the combination of AOH-AME, consisting of an increase in the percentage of ROS (+) cells and the oxidation of lipids, proteins, and DNA as compared with the individual toxin effect. A breakdown of the antioxidant defense was observed in IPEC-1 cells after the exposure to individual toxins, related to the decrease in the activity of antioxidant enzymes superoxide dismutase (SOD) and catalase (CAT), but no additive or synergic effect resulted after the exposure to the mixture of the toxins. In conclusion, our data indicate that both AOH and AME interfere with cell proliferation and oxidative stress. Moreover, the exposure of IPEC-1 cells to the combination of AOH and AME mycotoxins had a dose-dependent synergistic effect on IPEC-1 cell viability. Also, the oxidative damage induced in IPEC-1 cells by the combination of AOH and AME was stronger than the effects of individual toxins. However, the signaling pathways