Metabolism of 2‐acetylaminofluorene in primary rat hepatocyte cultures

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Abstract
Primary cultures of adult rat parenchymal hepatocytes were developed as an in vitro model to investigate the biochemical fate of the hepatocarcinogen 2-acetylaminofluorene (AAF). Viable cells were isolated by collagenase perfusion and cultured 2-5 days on collagen-coated dishes in serum-free culture medium containing hormones and other factors to retard the decline of cytochrome P-450. All of 137 ng or 13.7 .mu.g AAF was metabolized in 21-24 h by 2 .times. 106 cultured hepatocytes in 4.0 ml defined medium. At the higher dose, water-soluble metabolites appeared at 70% of the rate of metabolism at the lower dose, which was 17 ng/h for the initial 4 h. As the parent compound was consumed, bound AAF residues were recovered with hepatocellular macromolecules, accounting for a maximum of 5% of the 137-ng dose. Hormones in the culture medium stimulated the rate of appearance of water-soluble metabolites of AAF, correlating with the enhanced cytochrome P-450 levels of hormone-treated cells. Metabolism of AAF was diminished 50% during 3 h of incubation with 10-4 M SKF 525A [proadifen hydrochloride] and 100% with 10-3 M SKF 525A. At a dose of 40 .mu.g AAF per 2 .times. 106 cells, only 31% of the carcinogen was recovered from the culture medium as water-soluble products after 24 h; the cells were capable of metabolizing a subsequent 40-.mu.g dose at an undiminished rate, suggesting that saturation of metabolizing enzymes occurred. Primary hepatocyte cultures are a valid model system for quantitative investigations of the biochemical fate of AAF in mammalian cells.

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