Drug-induced alterations in the metabolome of primary hepatocytes

Background: Xenobiotics can induce rapid changes in the metabolome of cells even at non-toxic doses. This might result in the disturbance of metabolite and cell membrane compositions. The latter affects cellular signaling and transport processes. The effect of low, non-toxic doses of acetaminophen on the cell membrane composition was studied in primary hepatocytes, using ToF-SIMS. Methods: Primary rat hepatocytes were isolated using the collagenase perfusion technique and seeded on collagen-coated wafers. After treatment for 24h with different concentrations of acetaminophen cells were fast-frozen, freeze dried and the cell membrane composition was analyzed using ToF-SIMS. In parallel, the EC50 value of acetaminophen for the endpoint cytotoxicity was determined in these primary cells via MTT assay. Results: Incubation of rat hepatocytes revealed low toxicity of acetaminophen, with an EC50 value larger than 1 mM. Conversely, the application of ToF-SIMS demonstrated that metabolic changes were already detectable in the cell membrane in individual hepatocytes at 40 fold lower concentrations. Treatment with 2, 25, 100 and 500µM acetaminophen for 24h resulted in significant changes in the composition of the cell membrane. These changes were visualized using Fisher's discriminant analysis. The different treatment doses were clearly separated from each other and thus can be used as kind of “finger-prints”. Therefore we conclude that ToF-SIMS is capable of indicating changes in the metabolite pattern at cellular levels, which are already occurring at low, non-toxic concentrations of drugs and xenobiotics. Financial support through SFP 1322–530 is acknowledged.