Evaluation of the Cytotoxicity of Biochar Aqueous Extract in Caco-2 Cells: Time-Dependent Regulation of Apoptosis, Associated with miRNA Modulation

Biochar, a carbon-rich material traditionally used to improve soil health and as a feed additive, has recently attracted attention for its potential biological activity. This study investigated the effects of an aqueous biochar extract (BC-AE) on human intestinal epithelial cells (Caco-2), specifically examining its impact on cell viability and apoptosis. The metabolic activity of Caco-2 cells exposed to BC-AE was first evaluated using an MTS assay. A concentration of 3 mg/mL, which promoted Caco-2 metabolic activity, was selected for further testing at 24 and 72 hours. The effect of BC-AE on cell viability was assessed by epifluorescence microscopy (morphology) and flow cytometry (apoptosis profiling). The transcriptional response of cell viability-related genes (BAX, BAD, BCL-2, BCL-xL, MCL-1, P21, and P53) and microRNAs (miR-15b, miR-19, miR-21, miR-33a, miR-155, and miR-486) was analyzed by RT-qPCR. In parallel, selected proteins (BAD, BAX, BCL-2, and MCL-1) were examined by Western blotting. BC-AE decreased cell viability after 24 hours via late apoptosis, while 72-hour exposure increased necrosis without further viability loss. Both BAX and MCL-1 protein levels increased in Caco-2 cells after 72 hours of BC-AE treatment, and miR-15b and miR-21 were upregulated, suggesting the involvement of a regulatory mechanism controlling cell survival. The obtained findings highlight the importance of considering both concentration and exposure duration when assessing biochar bioactivity.