Hepatocyte primary cultures (HPC) derived from rat, mouse, hamster, and rabbit liver were characterized for a variety of parameters. The conditions that maximized recovery, attachment, and survival varied between species. Hepatocytes from all four species were capable of attaching in serum-free Williams' medium E (WME), but optimal attachment as monolayer cultures was achieved for mouse and hamster HPC in medium receiving 1% calf serum supplementation. Hamster hepatocytes required additional cations, whereas rabbit and rat hepatocytes displayed maximal attachment in medium supplemented with 10% calf serum. Survival of mouse and rabbit hepatocytes after 24 h in serum supplemented media was in the order of 90%. Rat and hamster hepatocyte 24 h survival was approximately 70 and 60%, respectively, and was not significantly affected by serum supplementation. Hepatocytes from each species varied in their content of cytochrome P450 at the time of isolation and in the rate of reduction during culture. Mouse and rat hepatocytes demonstrated the most rapid decline in content during the initial 24 h in culture, whereas concentrations in rabbit hepatocytes were virtually unchanged. The rate of decline in P450 concentrations in hamster hepatocytes was intermediate between those displayed by rat and rabbit hepatocytes. These studies have delineated conditions useful for the culture of hepatocytes from four species and have documented the status of an important parameter of their functional capability.
Genotoxicity was not elicited by any of a variety of organochlorine pesticides tested in the hepatocyte primary culture (HPC)/DNA repair assay utilizing hepatocytes from the rat, mouse, and hamster. DNA repair was observed in response to the positive control in all three systems. Thus the tumorigenicity of the organochlorine pesticides appears to reflect an epigenetic mechanism, probably involving a promotional effect resulting from a disruption in intercellular communication.
The relationship between acetylation rates of rabbit hepatocytes and their susceptibility to genotoxicity by DNAdamaging chemicals that undergo N-acetylation was studied in primary cultures of hepatocytes from New Zealand White rabbits that have a genetically determined difference in acetylation rates. Hepatocytes from rapid and slow acetylator rabbits maintained in culture the difference in acetylation rates that existed in viva DNA repair, an index of DNA damage, was produced by hydralazine in hepatocytes from slow acetylator rabbits but not in those from rapid acetylators. In contrast to these results, hepatocytes from rapid acetylators were more sensitive than those from slow acetylators to toxicity from the carcinogen 2-aminofluorene and displayed greater amounts of DNA repair. The amount of DNA repair measured with either chemical was dose dependent. These phenotype-dependent differences in the genotoxicity of two DNAdamaging chemicals provide evidence for the role of the acetylation polymorphism as a factor in determining susceptibility to toxicity, and perhaps carcinogenicity, of these chemicals. N-Acetylation rates of xenobiotics are under polymorphic genetic control in both humans and rabbits, resulting in individuals being either rapid or slow acetylators (1-5). This trait has been linked to toxicity and damage to DNA by chemicals of the aromatic amine or hydrazine type (6). For example, slow acetylator individuals are more likely than rapid acetylators to develop drug-related systemic lupus erythematosus (6-8). Individuals that develop this reaction have antinuclear antibodies as well as antibodies to DNA and nucleoproteins (6)(7)(8)(9)(10)(11)(12)(13). In vitro studies have also demonstrated interaction of systemic lupus erythematosus-inducing drugs with DNA (11,14,15).In the metabolism ofxenobiotics, N-acetylation is a step that can be followed by reactions such as N-hydroxylation and esterification, resulting in the generation of reactive metabolites that undergo covalent binding with cellular macromolecules, including DNA (16,17). Chemicals that can be acetylated and that also form covalent adducts with DNA include procainamide (18,19), isoniazid (20), and hydralazine (15), as well as the aromatic amine carcinogens, benzidine (21, 22), 2-aminofluorene, and 4-aminobiphenyl (23). Adduct formation by chemicals can be mediated by the enzymatic removal of the N-acetyl moiety (24,25), and evidence in the rabbit suggests that this reaction and the initial acetylation step are properties of the same enzyme (26).Because a difference in the acetylation rate can alter the proportion ofspecific metabolites that are formed (27), it is possible that genotoxicity-i. e., damage to DNA (28)-by substrates of N-acetyltransferase could be affected by the amount of acetylation. In order to investigate this possibility, we developed a model system that permitted measurement of both N-acetyltransferase activity and DNA damage in the same cells, using hepatocytes, which represent a major tissue of acetylation (...
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