Vanessa Partsch Vanessa Partsch Francesco Crudo Francesco Crudo Daniel Piller Daniel Piller Elisabeth Varga Elisabeth Varga Giorgia Del Favero Giorgia Del Favero Doris Marko Doris Marko Elsevier 2025 Alternaria mycotoxins may pose significant risks to human health due to their diverse spectrum of adverse effects and frequent occurrences in food. A previous study demonstrated the immunosuppressive, antiestrogenic, and genotoxic potential of a complex Alternaria mycotoxin extract (CE). The present study aimed to elucidate specific Alternaria mycotoxins or combinations thereof responsible for toxicity. Following toxicity-guided fractionation of the CE, a multiparametric panel of assays was applied to assess different endpoints. These included immunomodulatory effects (NF-?B reporter gene assay in THP1-Lucia™ monocytes), estrogenicity/antiestrogenicity (alkaline phosphatase assay in Ishikawa cells) and genotoxicity (?H2AX and alkaline comet assays in HepG2 cells). LC-MS/MS analysis revealed prominent mycotoxins in the active fractions, with altersetin (AST) identified as a novel key compound exhibiting immunoinhibitory (?2 ?M) and antiestrogenic (?5 ?M) properties in vitro. Additionally, while specific mycotoxin combinations explained the toxicity of active fractions, some effects remained unexplained, suggesting the presence of unidentified bioactive substances. This study underscores the significance of AST and specific toxin mixtures as major contributors to CE toxicity. Further, it highlights the importance of considering combinatory effects in risk assessment of Alternaria mycotoxins as well as further investigation of unknown Alternaria metabolites, which may pose additional health risks. PDFDocument eng 2025-04-10T09:28:45.947283Z 2025 Alternaria Humans Mycotoxins Toxicity Tandem Mass Spectrometry Hep G2 Cells Immunologic Factors Toxicity Immunologic Factors Pharmacology Estrogen Antagonists Toxicity Estrogen Antagonists Pharmacology DNA Damage Drug Effects Comet Assay 8056349 b application/pdf http://creativecommons.org/licenses/by/4.0/