Individual and combined effects of Aflatoxin B1, Deoxynivalenol and Zearalenone on HepG2 and RAW 264.7 cell lines

- Demonstrated the individual and combinatorial effect of commonly occurring mycotoxins- AFB1, DON and ZEN.
- Showed that, ranking based on cytotoxicity is DON > AFB1 > ZEN when tested individually.
- Showed that, binary (except for AFB1+ZEN) and tertiary combinations of mycotoxins leads to additive or synergistic effects.
- Demonstrated the utility of high content screening and mathematical modelling of data for elucidating combinatorial cytotoxicity of mycotoxins.

To understand the combinatorial toxicity of mycotoxins, we measured the effects of individual, binary and tertiary combinations of Aflatoxin B1 (AFB1), Deoxynivalenol (DON) and Zearalenone (ZEN) on the cell viability and cellular perturbations of HepG2 and RAW 264.7 cells. The nature of mycotoxins interactions was assessed using mathematical modeling (Chou-Talalay). Mechanisms of cytotoxicity were studied using high content screening (HCS) that probed cytotoxicity responses, such as changes in intracellular reactive oxygen species (ROS), mitochondrial membrane potential (MMP), intracellular calcium ([Ca2+]i) flux, and cell membrane damage. Our results showed that individual cytotoxicity of mycotoxins in a decreasing order was DON>AFB1>ZEN. Varying combinations of mycotoxins at differing concentrations showed different types of interactions. Most of the mixtures showed increasing toxic effects-synergism and/or addition while antagonistic effects were observed with combination of AFB1+ZEN. Generally, combination of mycotoxins showed significantly increased intracellular ROS production and [Ca2+]i flux, and decreased MMP in both cell lines, showing that the synergistic and additive effects of mycotoxin combination originate from perturbations of multiple cellular functions. Additionally, this study demonstrated the applicability of HCS for gaining mechanistic understanding on the toxicity of individual as well as combinatorial mycotoxins, and also provided scientific bases for formulating regulatory policies.