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 hepatocyt es to metabolize xenobiotics and steroids. As a first approach, the cy tochrome P450 content and markers of phase I and II metabolic reaction s were examined in subcellular fractions prepared from cultured liver cells. Ethoxyresorufin-O-deethylase (EROD) was chosen as a marker of C YP1A1. The induction of this enzyme was studied using beta-naphthoflav one 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 cell s was obtained by measuring selective hydroxylation of testosterone, a nd 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 gr adually until day 5 and then maintained on the same level (ca 50% of t he initial content) for 1 month in aggregate culture. EROD and GST act ivities 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-nitro phenol was used as substrate.