Mechanistic study of the cytotoxicity of cannabidiol and its metabolites in HepG2 cells.

The cannabidiol (CBD)-based drug, Epidiolex, received approval from the U.S. Food and Drug Administration (FDA) for treating seizures in certain childhood-onset epileptic disorders. CBD-associated liver toxicity is a serious side effect listed on the drug label. Our previous studies demonstrated cytotoxicity in primary human hepatocytes and HepG2 cells induced by CBD, 7-hydroxy-CBD, and 7-carboxy-CBD, with cell cycle disturbances, endoplasmic reticulum (ER) stress, and apoptosis identified as the underlying mechanisms. In this study, using a transcriptomic approach with mRNA-sequencing analysis, we found that downregulation of genes associated with oxidative phosphorylation and upregulation of genes associated with mitochondrial dysfunction, autophagy, and ER stress were among the top 10 canonical pathways consistently affected across different CBD concentrations. Direct measurement of the activity of the mitochondrial respiratory complexes that compose the oxidative phosphorylation process, demonstrated that CBD strongly inhibited Complexes IV and V and moderately inhibited Complexes II and III. CBD-induced mitochondrial dysfunction was indicated by a classic glucose-galactose assay, and the loss of mitochondrial membrane potential was confirmed by a JC-1 assay. Additionally, CBD induced autophagy, as evidenced by autophagosome formation and enhanced autophagic flux. Similar to CBD, 7-hydroxy-CBD induced a strong inhibition of Complexes IV and V, mitochondrial dysfunction, and autophagy, while 7-carboxy-CBD induced autophagy, with marginal inhibition on the respiratory complexes and no identified mitochondrial dysfunction. In summary, autophagy emerges as the common mechanism underlying CBD-, 7-hydroxy-CBD-, and 7-carboxy-CBD-induced cytotoxicity. Inhibition of mitochondrial respiratory complexes and mitochondrial dysfunction were observed with CBD and 7-hydroxy-CBD, but not with 7-carboxy-CBD.