Contributions of caspase-8 and -9 to liver injury from CYP2E1-produced metabolites of halogenated hydrocarbons

Abstract

1. Drug-induced liver injury is difficult to predict at the pre-clinical stage. This study aimed to clarify the roles of caspase-8 and -9 in CYP2E1 metabolite-induced liver injury in both rats and cell cultures in vitro treated with carbon tetrachloride (CCl₄), halothane or sevoflurane. The human hepatocarcinoma functional liver cell line was maintained in 3-dimensional culture alone or in co-culture with human acute monocytic leukemia cells.

2. In vivo, laboratory indices of liver dysfunction and histology were normal after administration of sevoflurane. CCl₄ treatment increased blood AST/ALT levels, liver caspase-3 and -9 activities and liver malondialdehyde, accompanied by centrilobular hepatocyte necrosis. Halothane increased AST/ALT levels, caspase-3 and -8 activities (but not malondialdehyde) concomitant with widespread hepatotoxicity. In vitro, CCl₄ treatment increased caspase-9 activity and decreased both mitochondrial membrane potential (MMP) and cell viability. In co-culture, halothane increased caspase-8 activity and decreased MMP and cellular viability. There were no toxic responses in CYP2E1 knockdown in monoculture and co-culture.

3. CYP2E1-inducing compounds play a pivotal role in halogenated hydrocarbon toxicity.

4. Changes in hepatocyte caspase-8 and -9 activities could be novel biomarkers of metabolites causing DILI, and in pre-clinical development of new pharmaceuticals can predict nascent DILI in the clinical stage.

Keywords:

• Caspase-8
• caspase-9
• CYP2E1 knockdown
• CYP2E1 metabolite-induced liver injury
• FLC-4
• three-dimensional culture

Acknowledgements

The authors are grateful to Professor Yoshinori Otsuki, Professor Masa-aki Shibata and Fuminori Takagi MS, for thoughtful comments and thank Maruishi Pharmaceutical Co., Ltd. for the gift of sevoflurane.

Declaration of interest

This work was supported in part by the TaNeDS program (DaiichiSankyo Co. Ltd.) and by Research Funds from Osaka University of Pharmaceutical Sciences.