Measurement of Xenobiotic Metabolising Enzyme Activities in Primary Monolayer Cultures of Immature Rainbow Trout Hepatocytes at Two Acclimatisation Temperatures

Abstract

Rainbow trout hepatocytes with a high viability were isolated by two-step collagenase perfusion through the portal vein. The yield was 1-2.5 x 106cells/g body weight. Culture conditions were defined for providing enhanced attachment and long-term cell survival at 7 ± 0.5°C and 15 ± 0.5°C, respectively. The hepatocytes, attached to Primaria™ plastic and cultured in Leibowitz L-15 medium with 9% fetal calf serum, were maintained as monolayers for 6–7 days. The activities in hepatocytes from immature trout of the biotransformation enzymes ethoxyresorufin-O-deethylase (EROD), aldrine epoxidase (AEPOX), NADPH-cytochrome c reductase (NCR), glutathione-S-transferase (GST) and UDP-glucuronosyltransferase (UDPGT), were all stable during the culture period. Differences in enzyme stability and activity (particularly the activity of EROD) between hepatocytes from different fish were observed at both temperatures. The temperature did not influence the activities of EROD or NCR, whereas AEPOX showed metabolic compensation. Both GST and UDPGT exhibited inverse temperature compensation. Hepatocyte monolayers, cultured from immature trout, may provide a useful system in pharmacological and toxicological research for investigating drug metabolism.