Immunomodulatory, Genotoxic, and Antiestrogenic Properties of the Alternaria Mycotoxins Tricycloalternarene 2b and Tricycloalternarene 3a

Abstract

Alternaria species produce a broad range of mycotoxins that frequently contaminate food and feed. While the toxicological properties of some Alternaria mycotoxins (e.g., alternariol, alternariol monomethyl ether, tenuazonic acid, and some perylene quinones) have been extensively investigated, toxicological information remains scarce for the majority of the Alternaria metabolites. In the present study, the two poorly characterized Alternaria mycotoxins tricycloalternarene 2b and tricycloalternarene 3a (TCA 2b and TCA 3a) were isolated from an Alternaria extract, structurally characterized by NMR spectroscopy, and evaluated with respect to their immunomodulatory, genotoxic, oxidative stress-inducing, and (anti)estrogenic properties. Both compounds were tested at concentrations ranging from 0.1 to 40 μM. Immunomodulatory effects were assessed using the nuclear factor κB (NF-κB) reporter gene assay in THP1-Lucia monocytes. Genotoxicity and intracellular oxidative stress were determined in HT-29 cells using the alkaline COMET assay in the absence and presence of formamidopyrimidine DNA glycosylase (FPG) and the DCF-DA assay, respectively. The ability of the compounds to interfere with the estrogenic pathway was investigated using the alkaline phosphatase (AlP) assay in Ishikawa cells. Both compounds suppressed lipopolysaccharide (LPS)-induced NF-κB pathway activation, with significant effects observed from 10 μM for TCA 2b and 20 μM for TCA 3a. Furthermore, both compounds induced concentration-dependent DNA damage, which was markedly enhanced by FPG treatment, resulting in significant DNA damage starting from 10 μM. In contrast, neither compound induced detectable intracellular oxidative stress under the applied experimental conditions. Of note, both compounds significantly reduced estradiol-induced AlP activity, indicating antiestrogenic effects at concentrations ≥20 μM for TCA 2b and ≥30 μM for TCA 3a. Overall, this study provides the first comprehensive toxicological characterization of TCA 2b and TCA 3a, and identifies both metabolites as biologically active Alternaria mycotoxins with immunosuppressive, genotoxic, and antiestrogenic properties in vitro.