title: Use of Primary Rat and Human Hepatocyte Sandwich Cultures for Activation of Indirect Carcinogens: Monitoring of DNA Strand Breaks and Gene Mutations in Co-cultured Cells.
abstract: Loss of cytochrome P-450 content is a common feature in conventional culture systems of primary hepatocytes. In contrast to the standard in vitro situation, in vivo each hepatocyte is exposed to an extracellular matrix (space of Disse) at two opposing basolateral surfaces. This in vivo symmetry has been reconstructed in vitro by culturing rat or human hepatocytes within two layers of collagen, thus forming a sandwich configuration. Activation of dimethylbenzanthracene (DMBA) or benzo[a]pyrene (BaP) was studied in rat and human hepatocytes. Genotoxic effects were studied in a three-dimensional co-culture model between sandwich hepatocytes and mammalian cells using the comet assay for detection of DNA strand breaks, and the HPRT test for detection of gene mutations. Sandwich hepatocytes generated active metabolites. The maintenance of metabolic properties in hepatocytes was dependent on extracellular matrix geometry. The number of DMBA- or BaP-induced genotoxic effects tended to be higher than in standard S-9 mix assays. While the ability to activate indirect carcinogens disappears within hours in primary hepatocytes, hepatocyte sandwich cultures enhance their ability to activate indirect carcinogens within 1 wk and retain this activity for up to 2 wk. This is the main advantage of the sandwich method over the more simple and conventional assays. While freshly isolated hepatocytes, regardless of whether in sandwich culture or in conventional assays, are injured by the isolation procedure and possess a corresponding reduced activation ability, hepatocytes in sandwich cultures recover over the course of a few days, and acquire a much higher ability to activate indirect carcinogens. Consequently, the indirect carcinogens BaP and DMBA, which were ineffective (BaP) or exhibited only weak effects (DMBA) at a concentration of 160nmol/ml in 1-2-day-old hepatocytes, were clearly effective (BaP) or showed about a threefold increase in genotoxicity (DMBA) in 8-day-old hepatocytes in sandwich co-culture. In contrast to the experiments with S-9 mix, which is toxic to mammalian cells and does not allow treatment times of more than 2-3hr, cells in co-culture with human or rat hepatocytes can be treated for at least 24hr. The use of sandwich cultures has not yet been described for genotoxicity studies. The results of the present study may perhaps facilitate the acceptance of this method as a co-culture model for the field of genetic toxicology. Use of hepatocytes alone for genotoxicity studies cannot be recommended for difficulties in isolating intact cells from the sandwich cultures. The use of human hepatocytes in sandwich co-culture should enable a more relevant evaluation of potential human genotoxicity with specific chemicals and should put the extrapolation of genetic toxicology data from animal species to humans on a more scientific basis. Beyond that, experiments with animals in vivo could be avoided.
authors.affiliation: Fraunhofer-Institute for Toxicology and Aerosol Research, Department of Genetics, Nikolai-Fuchs-Strasse 1, D-30625 Hannover, Germany.
authors.forename: R
authors.initials: R
authors.lastname: Fahrig
authors.forename: M
authors.initials: M
authors.lastname: Rupp
authors.forename: A
authors.initials: A
authors.lastname: Steinkamp-Zucht
authors.forename: A
authors.initials: A
authors.lastname: Bader