Maintenance of cytochrome P450 content and phase I and phase II enzyme activities in trout hepatocytes cultured as spheroidal aggregates

We investigated for 1 month the capacity of aggregated trout hepatocytes to metabolize xenobiotics and steroids. As a first approach, the cytochrome P450 content and markers of phase I and II metabolic reactions were examined in subcellular fractions prepared from cultured liver cells. Ethoxyresorufin-O-deethylase (EROD) was chosen as a marker of CYP1A1. The induction of this enzyme was studied using beta-naphthoflavone as inducer. For phase II reactions, glutathione-S-transferase (GST) activity was measured using 1-chloro-2,4-dinitrobenzene as substrate. A control of monooxygenase and transferase activities in intact cells was obtained by measuring selective hydroxylation of testosterone, and glucuronidation of 4-nitrophenol and testosterone. All the results were compared with those obtained for freshly isolated hepatocytes or conventional primary culture. The cytochrome P450 content decreased gradually until day 5 and then maintained on the same level (ca 50% of the initial content) for 1 month in aggregate culture. EROD and GST activities were constant or even increasing during 1 month. After 1 month culture, 2 days exposure of cells to beta-naphthoflavone led to a 10-fold increase. After 30 days, testosterone hydroxylase activities were about 30% of the activities found on freshly isolated hepatocytes. A similar decrease appeared for testosterone glucuronidation, whereas a stable UDPGT activity was observed during the same period when 4-nitrophenol was used as substrate.