Cellular mechanisms of the cytotoxic effects of the zearalenone metabolites α-zearalenol and β-zearalenol on RAW264.7 macrophages

Zearalenone (ZEN) and its metabolites are commonly found in many food commodities and are known to cause reproductive disorders and genotoxic effects. The major ZEN metabolites are α-zearalenol (α-ZOL) and β-zearalenol (β-ZOL). Although many studies have demonstrated the cytotoxic effects of these metabolites, the mechanisms by which α-ZOL or β-ZOL mediates their cytotoxic effects appear to differ according to cell type and the exposed toxins. We evaluated the toxicity of α-ZOL and β-ZOL on RAW264.7 macrophages and investigated the underlying mechanisms. β-ZOL not only more strongly reduced the viability of cells than did α-ZOL, but it also induced cell death mainly by apoptosis rather than necrosis. The ZEN metabolites induced loss of mitochondrial membrane potential (MMP), mitochondrial changes in Bcl-2 and Bax proteins, and cytoplasmic release of cytochrome c and apoptosis-inducing factor (AIF). Use of an inhibitor specific to c-Jun N-terminal kinase (JNK), p38 kinase or p53, but not pan-caspase or caspase-8, decreased the toxin-induced generation of reactive oxygen species (ROS) and also attenuated the α-ZOL- or β-ZOL-induced decrease of cell viability. Antioxidative enzyme or compounds such as catalase, acteoside, and (E)-1-(3,4-dihydroxyphenethyl)-3-(4-hydroxystyryl)urea suppressed the ZEN metabolite-mediated reduction of cell viability. Further, knockdown of AIF via siRNA transfection diminished the ZEN metabolite-induced cell death. Collectively, these results suggest that the activation of p53, JNK or p38 kinase by ZEN metabolites is the main upstream signal required for the mitochondrial alteration of Bcl-2/Bax signaling pathways and intracellular ROS generation, while MMP loss and nuclear translocation of AIF are the critical downstream events for ZEN metabolite-mediated apoptosis in macrophages.