Mechanistic insights into deoxynivalenol-induced cytotoxicity in an SH-SY5Y 3D spheroid model

Deoxynivalenol (DON) is a trichothecene mycotoxin frequently detected in cereal-derived foods and animal feed. Exposure to DON has been associated with a variety of toxic effects, including neurotoxicity. Three-dimensional (3D) cell culture systems have emerged as more physiologically relevant models than conventional two-dimensional (2D) cultures, as they better reproduce cellular organization, including cell-cell and cell-matrix interactions, and more accurately reflect in vivo conditions. In this context, the present study aimed to investigate the cytotoxic effects of DON and its underlying mechanisms using a 3D spheroid model derived from the human neuroblastoma cell line SH-SY5Y. Spheroids were exposed to different concentrations of DON and cell viability was assessed using the MTT assay, yielding an IC₅₀ value > 64 µM. No significant changes were detected in total intracellular reactive oxygen species (ROS) levels. However, a significant increase in mitochondrial superoxide production was observed at 16 µM, together with alterations in mitochondrial membrane potential between 2 and 16 µM. In addition, DON treatment altered cell cycle progression and increased the proportion of early apoptotic cells at 16 µM, as determined by Annexin V/propidium iodide staining. Protein analysis further revealed increase expression of Bax and Bcl-2 at 16 µM, resulting in a Bax/Bcl-2 ratio close to unity, along with elevated levels of cleaved caspase-3. Overall, these results demonstrate that DON induces cytotoxic effects in SH-SY5Y spheroids, accompanied by mitochondrial alterations, increased mitochondrial superoxide production, and apoptotic cell death. These findings contribute to the understanding of DON-induced alterations in neuronal cells and highlight the potential of SH-SY5Y spheroids as a biologically relevant human in vitro model for mechanistic studies of mycotoxin toxicity.