Use of a standardised and validated long-term human hepatocyte culture system for repetitive analyses of drugs: Repeated administrations of acetaminophen reduces albumin and urea secretion

Abstract

Human hepatocytes are the in vitro system of choice to study drug-induced processes in man. Here, we present HEPAC(2): a standardised and validated culture system in which human hepatocytes are maintained in HHMM (Human Hepatocyte Maintenance Medium) with HGF (hepatocyte growth factor) and EGF (epidermal growth factor). Cellular viability and hepatocellular functions were monitored daily. Albumin and urea production remained on a relatively constant level for up to 2-3 weeks. Based on this, a standard protocol was established that allows repeated exposure of hepatocytes to study drug metabolism. We used acetaminophen (AAP) to assay the feasibility of this system. Hepatocytes were exposed to AAP (100-2815 mg/l) for 24 h. Subsequently, the culture medium was replaced by medium without AAP and the same exposure scenario was repeated at intervals of 4 days. High doses of AAP (2815 mg/l) diminished urea production by 15-30% and albumin secretion by 70-80%. These effects were reversible. After removal of AAP, secretion of urea and albumin returned to control levels. AAP hepatotoxicity is caused by its biotransformation to the reactive metabolite N-acetyl-p-benzoquinoneimine (NAPQI) mediated by CYP2E1 and CYP1A2. The AAP activating enzymes were active for at least 21 days and their activity was maintained during at least four repeated cycles of exposure to AAP. In conclusion, these data demonstrate the suitability of our long-term culture system to serve as a tool for repetitive screening of drug-mediated changes of hepatocellular functions. This culture technique may help to overcome the sparse availability of human hepatocytes for testing drugmediated responses in man.