Combined in vitro and in silico mechanistic approach to explore the potential of Alternaria mycotoxins alternariol and altertoxin II to hamper γH2AX formation in DNA damage signaling pathways.

Risk assessment of food and environmental contaminants is faced by substantial data gaps and novel strategies are needed to support science-based regulatory actions. The Alternaria mycotoxins alternariol (AOH) and altertoxin II (ATXII) have garnered attention for their possible genotoxic effects. Nevertheless, data currently available are rather scattered, hindering a comprehensive hazard characterization. This study combined in vitro/in silico approaches to elucidate the potential of AOH and ATXII to induce double-strand breaks (DSBs) in HepG2 cells. Furthermore, it examines the impact of co-exposure to AOH and the DSB-inducing drug doxorubicin (Doxo) on γH2AX expression. AOH slightly increased γH2AX expression, whereas ATXII did not elicit this response. Interestingly, AOH suppressed Doxo-induced γH2AX expression, despite evidence of increased DNA damage in the comet assay. Building on these observations, AOH was postulated to inhibit γH2AX-forming kinases. Along this line, in silico analysis supported AOH potential interaction with the ATP-binding sites of these kinases and immunofluorescence experiments showed decreased intracellular phosphorylation events. Similarly, in silico results suggested that ATXII might also interact with these kinases. This study emphasizes the importance of understanding the implications of AOH-induced γH2AX expression inhibition on DNA repair processes and underscores the need for caution when interpreting γH2AX assay results.

Genotoxic and Mutagenic Effects of the Alternaria Mycotoxin Alternariol in Combination with the Process Contaminant Acrylamide.

Humans are constantly exposed to mixtures of different xenobiotics through their diet. One emerging concern is the Alternaria mycotoxin alternariol (AOH), which can occur in foods typically contaminated by the process contaminant acrylamide (AA). AA is a byproduct of the Maillard reaction produced in carbohydrate-rich foods during thermal processing. Given the genotoxic properties of AOH and AA as single compounds, as well as their potential co-occurrence in food, this study aimed to assess the cytotoxic, genotoxic, and mutagenic effects of these compounds in combination. Genotoxicity was assessed in HepG2 cells by quantifying the phosphorylation of the histone γ-H2AX, induced as a response to DNA double-strand breaks (DSBs). Mutagenicity was tested in Salmonella typhimurium strains TA98 and TA100 by applying the Ames microplate format test. Our results showed the ability of AOH and AA to induce DSBs and increase revertant numbers in S. typhimurium TA100, with AOH being more potent than AA. However, no synergistic effects were observed during the combined treatments. Notably, the results of the study suggest that the compounds exert mutagenic effects primarily through base pair substitutions. In summary, the data indicate no immediate cause for concern regarding synergistic health risks associated with the consumption of foods co-contaminated with AOH and AA.