Unpredicted synergistic genotoxic effects of co-occurring Alternaria mycotoxins at ratios representative of the European diet

Alternaria species are phytopathogenic fungi that contaminate a wide range of food commodities, including tomatoes, cereals and oilseeds. They simultaneously produce multiple mycotoxins, among which the most prevalent are alternariol (AOH), alternariol monomethyl ether (AME), tenuazonic acid (TeA), altenuene (ALT), and tentoxin (TEN). Their individual toxicological properties are well documented: AOH and AME are genotoxic, TeA is ribotoxic, whereas ALT and TEN exhibit low toxicity. Despite their frequent co-occurrence in food, the Thresholds of Toxicological Concern (TTC) established by the European Food Safety Authority (EFSA) do not take into account their combined effects which remain largely unexplored. This study provides the first evaluation of the genotoxic effects of these five co-occurring toxins in combination using rat intestinal IEC-6 cells exposed to mixtures reflecting European dietary ratios (AOH:AME:TeA:ALT:TEN = 1:1:15:1:1). AOH:AME mixtures exhibited synergistic genotoxicity, particularly at low concentrations. TeA, although non-genotoxic alone, markedly amplified DNA damage induced by AOH and AME, consistent with a mechanism involving impairment of protein synthesis due to ribotoxicity. ALT and TEN showed no intrinsic effect and did not modulate mixture genotoxicity. Cytotoxicity was mainly additive for AOH:AME mixtures but became synergistic in the presence of TeA. Overall, this study demonstrates for the first time that co-occurring Alternaria mycotoxins interact synergistically, revealing effects that cannot be predicted from individual compounds. These findings highlight the critical need to incorporate mixture effects into risk assessment frameworks.