Establishment of fish hepatocyte cultures for use in in vitro carcinogenicity studies

Methods were developed for the isolation and primary culture of rainbow trout (Salmo gairdneri) and channel catfish (Ictalurus punctatus) liver cells. Using a two-step perfusion technique, I isolated an average of 2.75 and 2.87 liver cells/g body weight from the trout and catfish, respectively. Hepatocytes represented 91.4% (trout) and 90.1% (catfish) of the total liver cells isolated. Both catfish and trout hepatocytes in primary culture displayed a linear decrease in survival with increased duration of culture. The DNA synthesis in the hepatocytes during culture showed a similar decrease with increased time in culture. Approximately 2.8% (trout) and 3.5% (catfish) of the hepatocytes exhibited nuclear labeling with [3H]dThd immediately after isolation. The labeling index decreased for hepatocytes from both species to 0% by the tenth day of culture. Activities of cytochrome P-450 and B-5 initially declined rapidly for both trout and catfish hepatocytes after placement in culture; however, these activities leveled off at low but measurable values for the first 8 days of culture. Unscheduled DNA synthesis (UDS) was induced in catfish and trout hepatocytes after exposure to dimethylnitrosamine, aflatoxin B1, benzo[a]pyrene, and N-methyl-N'-nitro-N-nitrosoguanidine. Trout hepatocytes displayed a decrease in UDS induction with aflatoxin B1 with increased age of the cultures. However, UDS induced by N-methyl-N'-nitro-N-nitrosoguanidine remained constant throughout the culture period.