The 32 P-post-labeling method in quantitative DNA adduct dosimetry of 2-acetylaminofluorene-induced mutagenicity in Chinese hamster ovary cells and Salmonella typhimurium TA1538

• Published in: Carcinogenesis (New York) 1987-04, Vol.8 (4), p.515-520
• Main Authors: Arce, Gail Theall, Dwain T., Cline, Mead, Janet E.
• Format: Article
• Language: English
• ISSN: 0143-3334; 1460-2180
• DOI: 10.1093/carcin/8.4.515

The usefulness of the 32 P-post-labeling/t.l.c. method for quantitative DNA adduct dosimetry was evaluated. 2-Acetylaminofluorene (2-AAF)-DNA adducts from three systems were characterized qualitatively and quantitatively by the 3 H-radiolabeled technique with subsequent analysis by h.p.l.c. (pre-labeling method) and by the 32 -post-labeling method. Both methods showed N -acetoxyacetylaminofluorene (N-OAc-AAF) reaction products with calf thymus DNA were predominantly N -(deoxyguanosm-8-yl)-2-acetylaminofhiorene (dG-C8-AAF) with some N -(deoxyguanosin-8-yl)-2-amino-fluorene (dG-C8-AF) and N -(deoxyguanosin- N  2 -yl)-2-acetyl-aminofluorene (dG-N2-AAF). In contrast, Chinese hamster ovary (CHO) cells treated with [ 3 H]N-OAc-AAF gave 80 or 90% dG-C8-AF adducts and 20 or 10% dG-C8-AAF adducts with the post- or pre-labeling method, respectively. Likewise in CHO cells treated with 2-AAF in the presence of rat liver homogenate, ˜90% dG-C8-AF and 10% dG-C8-AAF adducts were detected using the 32 P-post-labeling method. In Salmonella typhimurium strain TA1538 treated with 2-AAF or [ 3 H]2-AAF in the presence of a rat liver homogenate, one adduct, dG-C8-AF, was identified. Similar quantitative results were also obtained with the two methods. However, the 32 P-post-labeling method was more sensitive and also eliminated the use of radiolabeled-mutagen treatments. Quantitative DNA adduct dosimetry was applied to AAF-induced mutagenesis in the S. typhimurium and CHO/HPRT mutation assays. A linear and reproducible relationship existed between dG-C8-AF levels and AAF-induced mutants in both systems.