harmine 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

Dibenzofluoranthene-12,13-dihydrodiol (DBF-12,13-DHD) is six times more mutagenic in Salmonella TA100 than dibenzofluoranthene-3,4-dihydrodiol (DBF-3,4-DHD). However, these two major dibenzo[a,e]fluoranthene (DBF) proximate metabolites, which are immediate precursors of the corresponding diolepoxides, showed on an equimolar basis nearly identical initiation activities on mouse skin; they induced three times more papillomas than the parent hydrocarbon. On the other hand the epithelioma initiation capacities, i.e. the number of papillomas progressing to malignant tumours, of DBF or the two dibenzofluoranthene dihydrodiols were equivalent. Norharman, a putative vicinal diolepoxidation inhibitor in DBF metabolism when administered topically together with the initiation dose (100 nmol), strongly inhibited the induction of tumours by DBF-3,4-DHD and DBF. The relationship between in vitro mutagenic activity in Salmonella and the carcinogenicity of DBF metabolites in mice appears to be qualitative rather than quantitative.

Topics: Alkaloids; Animals; Carbolines; Carcinogens; Carcinoma; Female; Fluorenes; Harmine; Mice; Mice, Inbred Strains; Mutagenicity Tests; Mutagens; Mutation; Papilloma; Salmonella typhimurium; Skin Neoplasms; Structure-Activity Relationship

The production by dibenzo[a,e]fluoranthene (DBF) of DNA-protein cross-links in cultured mouse fibroblasts is probably mediated by the activation of proximate metabolites of DBF and not by the DBF molecule itself. In order to test this hypothesis, several agents that enhance or reduce production of the DBF metabolite putatively involved in cross-linking were tested. Increasing NADPH concentrations in the medium enhanced cross-link production; 1,2-epoxy-3,3,3-trichloropropane (TCPO), an inhibitor of epoxide hydrolases, slightly reduced DNA-protein cross-link formation at high concentrations; norharman (NH), an inhibitor of certain steps in the metabolism of DBF, totally blocked cross-linking. The possible involvement of DBF-bisdihydrodiol, a bifunctional metabolite identified in vitro, is discussed. Postincubation in DBF-free medium did not induce a significant reduction in cross-links, indicating that repair did not take place.

Topics: Animals; Carbolines; Cells, Cultured; DNA; Fibroblasts; Fluorenes; Harmine; Mice; NADP; Proteins; Trichloroepoxypropane

The structural identification of nineteen metabolites of dibenzo[a,e]fluoranthene (DBF) obtained by incubation in rat and mouse liver microsomes, allows one to establish a qualitative and semi-quantitative metabolic chart, involving up to three distinct oxidative attacks. The primary steps lead to dihydrodiols on rings A and D and phenols on rings A and E. Secondary vicinal epoxidation of dihydrodiols is a minor route as compared to attack at a second peripheral ring. Even after a third oxidation, one of the peripheral rings A, D and E remains unsubstituted. A model for cytochrome P-450 enzymatic activity which takes into account most of the observations is proposed. It requires that the catalytic site for monooxygenation is 0.6 nm apart from the center of an hydrophobic protein site accommodating one of the unsubstituted peripheral benzenoid rings. both trans diequatorial dihydrodiols of ring A and D corresponding to the 'bay' and 'pseudo bay region'; of DBF appear in the activation pathways for the in vivo carcinogenesis. The ultimate metabolite reacting with DNA is thus, most probably, a vicinal dihydrodiol epoxide of ring A or D. The great complexity of the metabolic chart of DBF as compared to other carcinogenic polycyclic aromatic hydrocarbons leaves also the possibility of sequential reactions at these two distinct sites of the molecule.

Topics: Animals; Biotransformation; Carbolines; Carcinogens; Chromatography, High Pressure Liquid; Female; Fluorenes; Harmine; Kinetics; Male; Mice; Microsomes, Liver; Rats; Species Specificity

The effects of norharman (NH) on the metabolism of dibenzo[a,e]-fluoranthene (DBF) and on its fixation on DNA, RNA and proteins have been studied in vitro by incubation with S-9 and microsomes from rats and mice. NH causes a decrease of the activity of microsome monooxygenases proportionally to its concentration but has no effect on the activity of NADPH P-450 reductase nor on that of epoxide hydrolase. Paradoxically, the amount of DBF hydrophobic metabolites and especially that of diols and phenols, increases in the incubation mixture in the presence of NH; this increase is independent of the presence of conjugation enzymes of cytosol. NH does not modify the covalent binding of DBF on the microsome RNA, conversely it decreases the binding of DBF on the DNA and on the proteins of the incubation mixture. This could partly explain the increase of DBF diols and phenols by an accumulative effect. Two higher homologs of NH: benzo[g]-beta carboline and benzo[i]-beta carboline, tested under the same conditions, proved inhibitory.

Topics: Alkaloids; Animals; Aryl Hydrocarbon Hydroxylases; Carbolines; Carcinogens; DNA; Epoxide Hydrolases; Fluorenes; Harmine; In Vitro Techniques; Mice; Microsomes, Liver; NADPH-Ferrihemoprotein Reductase; Rats; RNA