title: Use of precision cut human liver slices for studying the metabolism and genotoxic potential of xenobiotics by means of the (32)P-postlabelling technique: steps towards method validation using testosterone and 2-aminofluorene.
abstract: 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.
authors.affiliation: Research Laboratories, Schering AG, 13342 Berlin, Germany.
authors.forename: A
authors.initials: A
authors.lastname: Baumann
authors.forename: W
authors.initials: W
authors.lastname: Feser
authors.forename: P
authors.initials: P
authors.lastname: Cramer
authors.forename: R S
authors.initials: RS
authors.lastname: Kerdar
authors.forename: H
authors.initials: H
authors.lastname: Blode
authors.forename: J
authors.initials: J
authors.lastname: Korber
authors.forename: W
authors.initials: W
authors.lastname: Kuhnz