Development of an in vitro co-culture system to assess the DILI-related potential of drugs

Introduction: Drug-induced liver injury (DILI) is the main reason for the withdrawal of drugs from the pharmaceutical market due to unpredictable severe toxic side effects. DILI can not be sufficiently explained by the direct toxicity of a drug on hepatocytes but it also involves the interaction of hepatocytes with non-parenchymal cells (NPC). For the study of the effects of xenobiotics on liver metabolism, the co-culture of human hepatic cells with monocytic human THP-1 cells was established and acetaminophen (AAP) was used as a model compound. AAP is a famous analgesic whose reactive intermediate N-acetyl-p-benzochinonimine (NAPQI) leads to cell necrosis and/or apoptosis of hepatic cells. Necrotic hepatocytes release strong pro-inflammatory molecules such as DNA fragments and high mobility group box protein-1 (HMGBP1) which might activate hepatic NPC such as Kupffer cells. Here we investigated the toxic effects of AAP on THP-1 cells, cultured in combination with the hepatic cell lines Huh-7 or HepG2. In addition, the activity and the expression of several CYP450 enzymes in the cell lines were measured.

Results: The toxicity of AAP on THP-1 cells in the absence of hepatic cells was dependent on the stage of differentiation. In the co-culture of THP-1 with Huh-7 cells 10 mM AAP showed an increased toxicity, compared to the single culture. No increased toxicity was observed in the direct co-culture with HepG2 cells. In indirect co-culture models (incubation with inserts or application of the supernatant) no difference could be observed. Therefore it seems that THP-1 and hepatic cells had to be in direct contact to study the toxicity of the model drug AAP with sufficient sensitivity. Differences in the effects of AAP were related to differences in the expression of CYP450 mRNA and the enzyme activity. Whether the activity of tyrosinase, which also generates NAPQI, plays an additional role in the observed toxicity will be investigated next. In conclusion co-culture systems of human hepatic and monocytic cell lines seem to be suitable to study the DILI-related potential of hepatotoxic substances.