phaidra.vetmeduni.ac.at o:4028 DOI 10.1016/j.fct.2025.115315 Resolving complexity: identification of altersetin and toxin mixtures responsible for the immunomodulatory, antiestrogenic and genotoxic potential of a complex Alternaria mycotoxin extract 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. eng 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 2025-04-10T09:28:46.577Z LOMv1.0 Author BEGIN:VCARD VERSION:3.0 N:Partsch;Vanessa; FN:Vanessa Partsch END:VCARD BEGIN:VCARD VERSION:3.0 N:Crudo;Francesco; FN:Francesco Crudo END:VCARD BEGIN:VCARD VERSION:3.0 N:Piller;Daniel; FN:Daniel Piller END:VCARD BEGIN:VCARD VERSION:3.0 N:Varga;Elisabeth; FN:Elisabeth Varga X-ORCID:https://orcid.org/0000-0001-6046-3259 END:VCARD BEGIN:VCARD VERSION:3.0 N:Del Favero;Giorgia; FN:Giorgia Del Favero END:VCARD BEGIN:VCARD VERSION:3.0 N:Marko;Doris; FN:Doris Marko END:VCARD https://w3id.org/kim/hcrt/scheme https://w3id.org/kim/hcrt/text Textdokument Text LOMv1.0 yes https://creativecommons.org/licenses/by/4.0 application/pdf 8056349 https://phaidra.vetmeduni.ac.at/o:4028 https://phaidra.vetmeduni.ac.at/api/object/o:4028/thumbnail LOMv1.0 discipline