ATLA::Alternatives to Laboratory Animals

Volume 24, Number 5

Measurement of xenobiotic metabolising enzyme activities in primary monolayer cultures of immature rainbow trout hepatocytes at two acclimatisation temperatures.

ATLA 24, 727-740, September/October 1996

Elisabeth G. Jensen, Rune Thauland and Nils E. Søli

Department of Pharmacology, Microbiology and Food Hygiene, Norwegian College of Veterinary Medicine, P.O. Box 8146 Dept., 0033 Oslo, Norway

SUMMARY

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 106 cells/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 PrimariaTM plastic and cultured in Leibowitz L-15 medium with 97 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 (ERODE, 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.

Keywords: immature trout, hepatocytes, primary culture, cytochrome P45O-dependent mixed function oxidase, NADPH-cytochronze c reductase, conjugation enzymes, acclimatisation temperature