Establishment of a novel CYP3A4-transduced human hepatic sinusoidal endothelial cell model and its application in screening hepatotoxicity of pyrrolizidine alkaloids.

Abstract

Pyrrolizidine alkaloids (PAs) are extensively distributed in plants and are known to damage hepatic sinusoidal endothelial cells (HSECs) via metabolic activation mediated by hepatic cytochrome P450 enzymes (CYPs), particularly the CYP3A4 isozyme. Different PAs have distinct toxic potencies and their toxic effects on HSECs are difficult to be determined in cultured cells, because HSECs lack the key CYP3A4 isozyme for metabolic activation. This study aims to establish a novel, convenient and reliable CYP3A4-expressing HSEC model using human HSECs transduced with lentivirus carrying CYP3A4-ires-eGFP, for evaluating the hepatotoxicity of different PAs on their target HSECs. The developed CYP3A4-expressing HSEC (HSEC-CYP3A4) model was verified by the expression of GFP and CYP3A4 and by the ability to metabolize nifedipine, a classic CYP3A4 substrate. Treated with retrorsine, a representative toxic PA, HSEC-CYP3A4 cells showed significantly reduced cell viability, depletion of GSH, and increased formation of pyrrole-protein adducts. Furthermore, this newly developed cell model successfully discriminated the cytotoxic potency of different PAs evidenced by their IC₄₀ values. In conclusion, the established HSEC-CYP3A4 cell model can be used as a rapid screening platform for assessing the relative potencies of individual PAs on their target HSECs and for investigating the mechanisms underlying PA-induced hepatic sinusoidal damage.