Evaluation of amiodarone-induced phospholipidosis by in vitro system of 3D cultured rat hepatocytes in gel entrapment

Various mechanisms are involved in drug hepatotoxicity including reactive intermediates, steatosis, phospholipidosis, mitochondrial dysfunction, etc. Although 3D cultured hepatocytes reflected more CYP 450 mediated reactive intermediates than traditional 2D cultured hepatocytes, the 3D model has not been evaluated for drug-induced phospholipidosis. This study applied 3D cultured hepatocytes in gel entrapment for drug-induced phospholipidosis, and amiodarone was used as a model drug. By amiodarone exposure for 48 h, 3D cultured hepatocytes showed large number of lysosomal lamellar bodies (indicating phospholipidosis) and toxic response at a low dose of 2.5 μM, equivalent to toxic serum concentration in rats. This sensitivity to amiodarone-induced phospholipidosis might relate to the more intracellular drug distribution in 3D cultured hepatocytes. Moreover, steatosis, mitochondrial injury and oxidative stress were all sensitively detected in 3D cultured hepatocytes, well reflecting the involvement of these mechanisms in amiodarone hepatotoxicity. In addition, pretreatment of 3D cultured hepatocytes by CYP 3A1/2 inhibitor (ketoconazole) significantly reduced the toxicity of amiodarone, indicating the positive mediation of CYP 3A1/2. By comparison, 2D cultured hepatocytes did not show significant phospholipidosis and involvement of CYP 3A1/2. In conclusion, hepatocytes in 3D culture well reflected amiodarone toxicity and thus could be a promising model for phospholipidosis study.