Use of precision-cut human liver slices for studying the metabolism and genotoxic potential of xenobiotics by means of the P-32-postlabelling technique: steps towards method validation using testosterone and 2-aminofluorene

In the present study, a new in vitro model combining the short-term incubat ion of precision-cut human liver slices with DNA-adduct analysis by the P-3 2-postlabelling technique is proposed for investigation of the genotoxic po tential of xenobiotics. For method validation, the metabolic turnover of te stosterone (TES) and the DNA-adduct inducing potential of 2-aminofluorene ( 2-AF) were used. Precision-cur human liver slices were prepared from a tota l of 12 human liver samples which were freshly obtained as parts of resecta tes from liver surgery The slices were incubated as submersion cultures wit h TES and 2-AF for up to 6 h in 12-well tissue culture plates at concentrat ions of 10-50 and 0.06-28 mu M, respectively. Slices recovered from the sli cing procedure in the 4 degrees 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 metabo lized by human liver slices with a similar metabolite pattern as observed i n vivo. Almost 90% of the metabolites were conjugates. Major phase-I metabo lites a ere androstendione, 6 beta-OH-androsrendione, 6 beta-OH-TES, and 15 beta-OH-TES. After incubation with 2-AF, substance related DNA-adducts wer e detected which increased dose-dependently from 12 to 1146 adducts per 10( 9) nucleotides. The adduct pattern consisted of one major adduct spot, A, r epresenting 80-90% of the total adduct level and up to four minor adduct sp ots, B-E. In summary, the present data demonstrate that precision-cut liver slices are a valuable alternative in vitro system for DNA-adduct determina tion to screen chemicals for potential genotoxicity in humans.