Precision cut liver slices from humans and rats were used to investigate the covalent binding of xenobiotics to the DNA by means of the (32)P-postlabeling technique. Human liver slices were incubated with the structurally related steroid hormones chlormadinone acetate (5 mu g/ml), cyproterone acetate (0.01-5 mu g/ml), megestrol acetate (5 mu g/ml), and the positive control 2-aminofluorene (0.01-5 mu g/ml), which is known for its marked ability to form DNA-adducts in vivo. Rat liver slices were incubated with cyproterone acetate in concentrations of 0.1, 1, and 5 mu g/ml. The functional viability and metabolic activity of the slices were shown to be sufficiently maintained during the incubation time by measurement of the intracellular K(+)-content and the metabolic turnover of the model substrate 7-ethoxycoumarin, respectively. All three test substances and the control induced DNA-adducts in human liver slices, however, with a different adduct pattern. While the total DNA-adduct levels obtained with cyproterone acetate and megestrol acetate were in the same order of magnitude (on average 1000 DNA-adducts/10(9) nucleotides after incubation with 5 mu g /ml), the relative adduct labeling calculated for chlormadinone acetate was about 400. Following in vitro incubation of rat liver slices with cyproterone acetate, the relative adduct labeling values increased proportionally with increasing concentrations and added linearily to in vivo generated DNA-adducts. At the level of liver slices, different DNA-adduct patterns were induced by cyproterone acetate in rat and man. In contrast to the finding of others, using rat hepatocytes, the relative adduct labeling values of cyproterone acetate and megestrol acetate were in the same order of magnitude after incubation with human liver slices. The present study indicates that liver slices are a useful tool to investigate the in vitro DNA-adduct inducing potential of xenobiotics.

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In the present study, a new in vitro model combining the short-term incubation of precision-cut human liver slices with DNA-adduct analysis by the (32)P-postlabelling technique is proposed for investigation of the genotoxic potential of xenobiotics. For method validation, the metabolic turnover of testosterone (TES) and the DNA-adduct inducing potential of 2-aminofluorene (2-AF) were used. Precision-cut human liver slices were prepared from a total of 12 human liver samples which were freshly obtained as parts of resectates from liver surgery. The slices were incubated as submersion cultures with TES and 2-AF for up to 6 h in 12-well tissue culture plates at concentrations of 10-50 and 0.06-28 μM, respectively. Slices recovered from the slicing procedure in the 4 °C cold Krebs-Henseleit buffer as indicated by intracellular potassium concentrations which increased for 2 h and then remained stable until the end of the incubation. TES was extensively metabolized by human liver slices with a similar metabolite pattern as observed in vivo. Almost 90% of the metabolites were conjugates. Major phase-I metabolites were androstendione, 6β-OH-androstendione, 6β-OH-TES, and 15β-OHTES. After incubation with 2-AF, substance related DNA-adducts were detected which increased dose-dependently from 12 to 1146 adducts per 10(9) nucleotides. The adduct pattern consisted of one major adduct spot, A, representing 80-90% of the total adduct level and up to four minor adduct spots, B-E. In summary, the present data demonstrate that precision-cut liver slices are a valuable alternative in vitro system for DNA-adduct determination to screen chemicals for potential genotoxicity in humans.