Abstract

Aims 3- n -Butylphthalide (NBP) is widely used for the treatment of cerebral ischaemic stroke but can causeliver injury in clinical practice. This study aims to elucidate the underlying mechanisms and propose potential preventive strategies. Main methods NBP and its four major metabolites, 3-hydroxy-NBP (3-OH-NBP), 10-hydroxy-NBP, 10-keto-NBP and NBP-11-oic acid, were synthesized and evaluated in primary human or rat hepatocytes (PHHs, PRHs). NBP-related substances or amino acid adducts were identified and semi-quantitated by ultra-high performance liquid chromatography coupled to high-resolution mass spectrometry (UHPLC-HRMS). The target proteins and binding sites were identified by shotgun proteomics based on peptide mass fingerprinting coupled with tandem mass spectrometry and verified by molecular docking. Key findings The toxicity of NBP and its four major metabolites were compared in both PHHs and PRHs, and 3-OH-NBP was found to be the most toxic metabolite. 3-OH-NBP induced remarkable cell death and oxidative stresses in hepatocytes, which correlated well with the levels of glutathione and N -acetylcysteine adducts (3-GSH-NBP and 3-NAC-NBP) in cell supernatants. Additionally, 3-OH-NBP covalently conjugated with intracellular Cys, Lys and Ser, with preferable binding to Cys sites at Myh9 Cys1380, Prdx4 Cys53, Vdac2 Cys48 and Vdac3 Cys36. Furthermore, we found that CYP3A4 induction by rifampicin augmented NBP-induced cell toxicity and supplementing with GSH or NAC alleviated the oxidative stresses and reactive metabolites caused by 3-OH-NBP. Significance Our work suggests that glutathione depletion, mitochondrial injury and covalent protein modification are the main causes of NBP-induced hepatotoxicity, which may be prevented by exogenous GSH or NAC supplementation and avoiding concomitant use of CYP3A4 inducers.