Toxicity evaluation of individual and mixed enniatins using an in vitro method with CHO-K1 cells

Enniatins (ENs) A, A1, B and B1 are produced by Fusarium species. They are known as emerging fusariotoxins, and can cause outbreaks in both humans and animals. ENs elicits a wide range of different biological properties and toxicological effects, and their co-occurrence may enhance the extent of these hazards. As the potential toxins reach in vitro cells in the same way as they would in vivo, cytotoxicity was studied with CHO-K1, which is considered one of the most sensitive cell lines for preliminary screening of cytotoxicity studies. In this study, individual cytotoxic effects of ENs were evaluated by MTT assay after exposing ENs to CHO-K1 cells for 24, 48, and 72h. The IC50 values obtained for ENs A, A1, B and B1 ranged from >7.5 to 2.83±0.49, from 8.8±2.29 to 1.65±0.06, from 11.0±2.65 to 2.80±0.16 and from 4.53±1.23 to 2.47±0.29μM, respectively. The ENs cytotoxic interaction was evaluated by the isobologram method. The IC50 values for ENs combination ranged from 0.44±0.15 (ENs A1+B1) to 0.97±0.48 (ENs A1+B+B1). The binary combinations ENs A+B1, ENs A1+B and ENs B+B1 showed additive effect thought all concentrations tested. Sinergistic effect of combined ENs A+A1, A+B, A1+B1, A+A1+B, A+A1+B1, A+B+B1 and A1+B+B1 at higher concentrations occurred. Synergism effect (CI from 0.37±0.08 to 0.74±0.22) was observed at higher concentrations with binary and tertiary combinations of EN A, while antagonism effect was obtained at lower concentrations for ENs A+A1+B1 (CI=2.67±1.32) and ENs A1+B+B1 (CI=3.14±1.91). These results provide quantitative evidence that ENs cytotoxicity depend on their concentrations, and also on their combination with other mycotoxins.