Phase I and II enzyme characterization of two sources of HepG2 cell lines

1. The metabolism by HepG2 cell from two sources (M1, M2) of 12 substrates is reported: ethoxyresorufin, ethoxycoumarin, testosterone, tolbutamide, chlorzoxazone, dextromethorphan, phenacetin, midazolam, acetaminophen, hydroxycoumarin, p-nitrophenol and 1-chloro-2,4-dinitrobenzene (CDNB), and a pharmaceutical compound, EMD68843. 2. Activities varied markedly. Some were present in M1 (CYP1A, CYP2C9, CYP2E1) but absent in M2. M1 had a more complete set of Phase I enzymes than M2. CYP1A2, CYP2C9, CYP2D6, CYP2E1 and CYP3A activities were present at levels similar to human hepatocytes. Phase II metabolism differed between M1 and M2. M1 conjugated hydroxycoumarin and p-nitrophenol to glucuronides only, whereas M2 produced sulfates. Glutathione conjugation of CDNB metabolism was 10-fold higher in M1 than in M2, but was still much lower than in human hepatocytes. CYP2E, CYP2C, CYP2B6 and CYP3A (but not CYP1A, glucuronyl S-transferase or S-transferase) were inducible in M1. Metabolites of EMD68843, produced by induced (but not uninduced) M1 were the same as those produced in human hepatocytes. 3. In conclusion, HepG2 cells have both Phase I and II enzymes, which activities and at what levels depend on the source and culture conditions. Therefore, HepG2 cells routinely used in in vitro assays should be characterized for their drug-metabolizing capabilities before any results can be fully interpreted.