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

Danica den Hollander; Celestien Holvoet; Kristel Demeyere; Noémie De Zutter; Kris Audenaert; Evelyne Meyer; Siska Croubels

doi:10.3389/fmicb.2022.849243

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

Front Microbiol. 2022 Apr 22:13:849243. doi: 10.3389/fmicb.2022.849243. eCollection 2022.

Authors

Danica den Hollander¹, Celestien Holvoet², Kristel Demeyere², Noémie De Zutter³, Kris Audenaert³, Evelyne Meyer², Siska Croubels¹

Affiliations

¹ Laboratory of Pharmacology and Toxicology, Department of Pathobiology, Pharmacology and Zoological Medicine, Faculty of Veterinary Medicine, Ghent University, Merelbeke, Belgium.
² Laboratory of Biochemistry, Department of Veterinary and Biosciences, Faculty of Veterinary Medicine, Ghent University, Merelbeke, Belgium.
³ Laboratory of Applied Mycology and Phenomics, Department of Plants and Crops, Faculty of Bioscience Engineering, Ghent University, Ghent, Belgium.

Abstract

Alternariol (AOH), alternariol monomethyl-ether (AME), and tenuazonic acid (TeA) are major mycotoxins produced by fungi of the genus Alternaria and are common contaminants of food products such as fruits, vegetables, cereals and grains. Alternaria 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 Alternaria mycotoxins in humans and livestock are generally lacking, precluding proper hazard characterization. This study aimed to fill some knowledge gaps by studying the in vitro 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.

Keywords: Caco-2 cells; HepG2 cells; alternariol; alternariol monomethyl-ether; binary and ternary combinations; cytotoxicity; flow cytometry; tenuazonic acid.