phosphorus-radioisotopes and dibenzo(a-e)fluoranthene

Quantitative and qualitative changes in the inhibition of DNA adduct formation in the presence of increasing concentrations of norharman (NH) were investigated in vivo in mouse fibroblasts treated with dibenzo[a,e]fluoranthene (DBF), a potent carcinogen in mice. The nuclease P1 modification of the 32P-postlabeling technique was used to identify adducts. A dose-dependent reduction in DBF-DNA adduct formation was observed: an 80% reduction with 0.06 mM NH and 90% with 0.12 mM NH. At 0.12 mM NH, all of the spots coming from hydroxylated DBF vicinal dihydrodiol (DHD) epoxides were missing; the only clear spot was that of the major DBF adduct produced by the ultimate DBF metabolite, DBF-3,4-DHD-1,2 oxide. Spots representing other DBF-DHD epoxide adducts appeared only in trace amounts. These results can be interpreted as a dose-dependent competition or inhibition of some secondary metabolic step, most probably secondary epoxidation; however, a direct protective effect of NH during adduct formation cannot be excluded. NH is a strong inhibitor of DBF-DNA adduct formation in vivo.

Topics: Animals; Carbolines; Carcinogens; Cells, Cultured; DNA; Female; Fibroblasts; Fluorenes; Harmine; Mice; Phosphorus Radioisotopes; Pregnancy

The formation of DNA adducts was investigated in mouse fibroblasts from two different tissues--embryos and adult lung--after incubation with dibenzo[a,e]fluoranthene (DBF) or its major proximate metabolites. The nuclease P1 modification of the 32P-postlabeling method was adapted for detection of DBF-DNA adducts. Quantitative and qualitative differences were observed in the metabolic activation mediated by the two cell types. DBF-DNA adducts generated three major spots reproducibly, and more than ten spots of medium or weak importance. The highest level of DNA binding occurred via the DBF-bay region vicinal dihydrodiol epoxide but with significant differences in the quantitative distribution of adducts. Striking qualitative differences were observed when lung fibroblasts were incubated with the DBF-pseudo bay region dihydrodiol (DBF-12,13-DHD). The spots representing adducts induced in embryo fibroblasts by DBF-3OH-12,13-DHD, a further metabolite of DBF-12,13-DHD, were totally absent from chromatograms of lung cells. These results show that both embryo and lung fibroblasts can activate DBF but that different cytochrome P-450 forms and substrate affinities are involved. The finding that different activation systems may be present in subcategories of the same tissue, may provide a partial explanation for the wide variations in sensitivity to carcinogens among species, organs and tissues.

Topics: Animals; Biotransformation; Carcinogens; Cells, Cultured; Dimethyl Sulfoxide; DNA; Embryo, Mammalian; Fibroblasts; Fluorenes; Lung; Mice; Mutagens; Oxygenases; Phosphorus Radioisotopes

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 polycyclic 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 greater than 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 dihydrodiol (+/- -trans-3,4-dihydro-3,4-dihydroxyDBF; DBF-3,4-DHD). Incubation with the DBF pseudo-bay region dihydrodiol (+/- -trans-12,13-dihydro-12,13-dihydroxyDBF; 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-pseudo-bay 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.

Topics: Animals; Carcinogens; Cells, Cultured; DNA; Fibroblasts; Fluorenes; Mice; Phosphorus Radioisotopes