P-32 POST-LABELING ANALYSIS OF DNA ADDUCTS IN MOUSE EMBRYO FIBROBLASTS TREATED WITH DIBENZO[A,E]FLUORANTHENE AND ITS MAJOR METABOLITES

The formation of DNA adducts was investigated in mouse fibroblasts treated with dibenzo [a, e] fluoranthene (DBF), using the nuclease P1 modification of the 32P-post-labeling method. In order to separate the poorly soluble, bulky DNA adducts of this potent sarcomogenic, six-ring poly cyclic aromatic hydrocarbon, several modifications of the method were introduced. Chromatographic spots were identified by incubating fibroblasts with the four major proximate metabolites of DBF and observing the co-migration of adducts with those of DBF. DNA-DBF adducts chromatographed very reproducibly in three major spots and in > 10 spots of medium or low importance. The most prominent spots, 2 and 3, were present characteristically after incubation of cells with the DBF-bay region dihydrodial (±-trans-3, 4-dihydro-3, 4-dihydroxy DBF; DBF-3, 4-DHD). Incubation with the DBF pseudo-bay region dihydrodiol (±-trans-12, 13-dihydro 12, 13-dihydroxy DBF; DBF-12, 13-DHD) gave rise to a more complex pattern of nine spots, two of which, spots 4 and 5, were prominent. Direct in vitro reaction between DNA and the synthetic anti-isomer of the DBF-bay region DHD epoxide yielded adducts in spots 2 and 3, while the DBF-anti-pseudobay region DHD epoxide yielded adducts in spots 4 and 5. Peripheral, fast-migrating spots present in the DBF chromatogram were identified as adducts of DBF-7OH-3, 4-DHD and DBF-3OH-12, 13-DHD. Major spot 1 was present in all DBF chromatograms but not after incubation with the DBF bay and pseudo-bay region proximate metabolites. Its probable origin as a non-bay region epoxide reaction is discussed. In previous experiments, the physicochemically very similar DBF-bay region and pseudo-bay region tritium-labeled adducts co-eluted in HPLC as a single peak. 32P-Post-labeling analysis allowed reproducible separation of DBF-DNA adducts and showed in addition the existence of several new adducts models of DBF. Quantification of DBF adducts made it possible to identify the DBF-bay region DHD epoxide and the metabolites responsible for spot 1 adducts as the major ultimate DBF metabolites in fibroblasts. © 1990 Oxford University Press.