An in vitro system for the identification of DILI related signaling?

Chemicals present in consumer products or drugs might pose a potential hazard to humans. The conversion of these chemicals into reactive intermediates by cellular metabolism may lead to the production of reactive oxygen species (ROS) and the formation of DNA or protein adducts. At low, non-toxic doses, Drug-Induced Liver Injury (DILI) can occur in rare incidence. It involves the immune system and often results in the withdrawal of drugs from the market once it becomes apparent. Here we investigated the toxic effects of the DILI-inducing drug, ketoconazole, on hepatocytes in vitro.

Ketoconazole, a drug used to treat fungal infections, is metabolized by CYP1A1. In July 2013 the European Medicines Agency recommended suspension of marketing authorization for oral ketoconazole as it can induce DILI by as yet unknown mechanisms.

Two hepatoma cell lines (HepG2 and Huh-7) and murine primary cells were cultivated and incubated with different concentrations of ketoconazole for 24h. Viability was quantified using the MTT and LDH assay while ROS were measured by 2',7'-dichlorodihydrofluorescein diacetate (H2DCFDA). Expression and concentration of IL-8, an important chemokine, were measured by qPCR and ELISA.

In human hepatoma cell lines as well as in primary cells ketoconazole induced a concentration-dependent generation of ROS (EC50 of 42 – 53µM), which is an important prerequsite for DILI induction. The CYP1A1 expression was increased under treatment conditions, thus indicating an activated metabolism of ketoconazole. Although no increase of IL-8 mRNA expression was detected, the concentration of IL8 was found well elevated in the supernatant though. Here we could show that ketoconazole induces ROS formation, CYP1A1 induction and IL-8 release in human hepatoma cell lines in vitro. In future experiments the expression levels of additional cytokines like TNF-α or IL-1 will be quantified in order to elucidate the underlying signaling pathways.

Corresponding author: Wewering, Franziska

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