Cytotoxic Effects of Alternariol, Alternariol Monomethyl-Ether, and Tenuazonic Acid and Their Relevant Combined Mixtures on Human Enterocytes and Hepatocytes

Abstract

Alternariol (AOH), alternariol monomethyl-ether (AME), and tenuazonic acid (TeA) are major mycotoxins produced by fungi of the genus <i>Alternaria</i> and are common contaminants of food products such as fruits, vegetables, cereals and grains. <i>Alternaria</i> mycotoxins are known to cause relevant economic losses and to have a negative impact on human and animal health. EFSA stated in its scientific opinion that data on the toxicity of <i>Alternaria</i> mycotoxins in humans and livestock are generally lacking, precluding proper hazard characterization. This study aimed to fill some knowledge gaps by studying the <i>in vitro</i> cytotoxicity toward human intestinal epithelial cells (Caco-2) and hepatocytes (HepG2). Cytotoxic properties were assessed by flow cytometric analyses of remaining viable cells (i.e., propidium iodide negative) after mycotoxin exposure for 24-48 h versus solvent control. Treatment of cells with single doses of AOH, AME, and TeA resulted in a dose-dependent loss of cell viability for both cell lines. Half maximal effective concentrations (EC₅₀) of the different mycotoxins were comparable for the two cell lines. On HepG2 cells, EC₅₀ values varying between 8 and 16, 4 and 5, and 40 and 95 μg/mL were calculated for AOH, AME, and TeA, respectively. On Caco-2 cells, EC₅₀ values of 19 μg/mL and varying between 6 and 23, and 60 and 90 μg/mL were calculated for AOH, AME, and TeA, respectively. A general relative cytotoxicity ranking of about 1 = 1 >>> 3 was obtained for AOH, AME, and TeA, respectively. Treatment of both cell lines with combined binary and ternary mixtures of AOH, AME, and TeA in a 1:1:3 ratio, also showed a dose-dependent decrease in cell viability. For both cell lines, the binary combination of especially AME and TeA (1:3 ratio) but also of AOH and AME (1:1 ratio) significantly increased the cytotoxicity compared to the single compound toxicity, although mainly at the highest concentrations tested. The ternary combinations of AOH, AME, and TeA induced only a slight increase in cytotoxicity compared to the single mycotoxins, again at the highest concentrations tested.