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

## TL;DR

This study shows that biochar extract affects intestinal cells by changing cell survival and death processes, with effects depending on exposure time.

## Contribution

The study reveals time-dependent regulation of apoptosis and miRNA modulation by biochar extract in Caco-2 cells.

## Key findings

- BC-AE decreased cell viability after 24 h via late apoptosis.
- 72 h exposure increased necrosis without further viability loss.
- miR-15b and miR-21 were upregulated, suggesting a regulatory mechanism for cell survival.

## Abstract

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 examined the effects of an aqueous biochar extract (BC-AE) on human intestinal epithelial cells (Caco-2), focusing on its influence 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 h. 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. We showed that BC-AE decreased cell viability after 24 h via late apoptosis, while 72 h exposure increased necrosis without further viability loss. Both BAX and MCL-1 protein levels increased in Caco-2 cells after 72 h 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 and represent an additional contribution to the ongoing effort to better understand the biological role.