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

被引:1
作者
Baumann, A
Feser, W
Cramer, P
Kerdar, RS
Blode, H
Körber, J
Kuhnz, W
机构
[1] Schering AG, Res Labs, D-13342 Berlin, Germany
[2] Humboldt Univ, Virchow Hosp, D-13353 Berlin, Germany
关键词
human liver slice; genotoxicity; DNA-adduct; P-32-postlabelling assay; metabolism;
D O I
10.1080/135475099230868
中图分类号
Q81 [生物工程学(生物技术)]; Q93 [微生物学];
学科分类号
071005 ; 0836 ; 090102 ; 100705 ;
摘要
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 P-32-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-cur 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 mu M, respectively. Slices recovered from the slicing 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 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 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 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.
引用
收藏
页码:188 / 202
页数:15
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