Comparative Cytotoxicity of DON and 3-keto-DON

The in vitro toxicity of DON and its oxidation product (3-keto-DON) were investigated using GES-1 cells and a CKK-8 test. GES-1 cells were bought from Cobioer Biosciences Co., Ltd. (Nanjing, China), and maintained under the cultivation conditions reported by Yang et al. [50 (link)]. First, 10,000 cells/well were plated into 96-well plates and allowed to proliferate to 80% confluence before cells were collected and exposed to 3-keto-DON (0.5, 1, 3, 6, 12, 24, and 48 mg/L) and DON standard (0.5, 1, 2, 4, 8, 10, and 20 mg/L) for 24 h. Following that, the spent medium was exchanged with a fresh one containing 1 mg/mL of CCK-8, which was then incubated for 4 h. Finally, cell viability was determined using an ELISA reader to measure the optical density at 450 nm. The percentage inhibition compared to control-treated cells was calculated for each compound concentration. All analyses were performed in triplicate. Statistical differences were determined using one-way ANOVA, with p < 0.05 considered to be statistically significant. IC₅₀ values were calculated using Dr Fit software version 1.042 [61 (link)] with default setting parameters.