Tissue-specific differences in adduct formation by hepatocarcinogenic and sarcomatogenic derivatives of 7H-dibenzo[c,g]carbazole in mouse parenchymal and nonparenchymal liver cells.

Parenchymal (PC) and nonparenchymal (NPC) liver cells have different tissue-specific, procarcinogen activation enzymes. NPC appear to be protected against the mutagenic effects of lipotropic bulky adducts induced by carcinogens by a unknown mechanism. Most studies of activation have been conducted with whole liver. The purpose of this study was to differentiate adduct formation in mouse PC and in NPC, isolated after in vivo administration of 7H-dibenzo(c,g)carbazole (DBC), the most efficient liver carcinogen in mice, which also has potent sarcomagenic and epitheliomagenic activities. The very sensitive 32P-postlabeling method was used to detect adducts. Two tissue-specific DBC derivatives, 6-methoxy-DBC (6MeODBC), which is exclusively sarcomagenic, and 5,9-dimethyl-DBC (DiMeDBC), which is exclusively hepatocarcinogenic, were analyzed in parallel. Both PC and NPC generated the ultimate metabolites of DBC, but NPC were substantially less efficient. Clear-cut tissue-specific differences in adduct formation were established: the sarcomagenic 6MeODBC gave rise only to NPC-DNA adducts, and the hepatocarcinogenic DiMeDBC only to PC-DNA adducts. The chromatograms of the adducts were compared with those of mouse embryonic cells in culture and mouse epidermal cells. The results are discussed in connection with animal experiments with DBC, 6MeODBC, and dimethylbenzanthracene and with published data on PC and NPC activating enzymes.

Interaction of 7H-dibenzo[c,g]carbazole and its organspecific derivatives with hepatic mitochondrial and nuclear DNA in the mouse.

The recent observation of a high level of adducts in mitochondrial DNA (mtDNA) of cells exposed to chemical carcinogens aroused new interest in the hypothesis that carcinogen-induced damage in mitochondria plays a role in one or more stages of carcinogenesis. In order to investigate whether differences in the metabolic activation of carcinogens have qualitative and quantitative effects on ml- and nuclear DNA (nuDNA) adduct formation, mice were exposed to the potent hepatocarcinogenic and sarcomagenic polycyclic hydrocarbon 7H-dibenzo[c,g]carbazole (DBC) and to three of its derivatives that show large differences in enzymatic activation: N-acetyl-DBC (N-AcDBC), which is carcinogenic for several tissues; 5,9-dimethyl-DBC (DiMeDBC), which is exclusively hepatocarcinogenic; and N-methyl-DBC (N-MeDBC), which is exclusively sarcomagenic. Adduct formation and toxic effects were measured over 48 hr. With a moderate 5 mumol/kg dose of DBC, the adduct level in liver 24 hr after treatment was always higher in nuDNA than in mtDNA; after 48 hr a substantial increase in the level of adducts in mtDNA was observed, with a parallel decrease in the level in nuDNA. With DiMeDBC, a 4.9-fold increase in mtDNA was seen at 48 hr, whereas, at the same dose, the non-hepatocarcinogenic N-MeDBC induced a very small number of adducts. In order to obtain a nearly identical level of adducts in nu- and mtDNA at 24 hr, the dose of DBC must be three times higher (15 mumol/kg); this and higher dose levels had a strong cytotoxic effect in liver cells. Qualitative differences in adduct distribution were observed on chromatograms of mtDNA and nuDNA, showing that the access to mtDNA is a complex process. Our results confirm that mouse liver mtDNA is a major target for DBC and its hepatocarcinogenic derivatives. The possible interference of genotoxic alterations in mtDNA with carcinogenic mechanisms is discussed.

Comparison of 7,12-dimethylbenz(a)anthracene-DNA adduction in the epidermis of two lines of mice selected for resistance (CAR-R) or susceptibility (CAR-S) to skin carcinogenesis.

Two lines of mice were produced by bidirectional selective breeding: one resistant (CAR-R) and one susceptible (CAR-S) to two-stage skin carcinogenesis by dimethylbenz(a)anthracene and 12-O-tetradecanoyl-phorbol-13-acetate. The dimethylbenz(a)anthracene-DNA adduct formation was compared in the two lines by a postlabeling procedure so as to determine whether the striking interline difference observed as to tumor incidence could (in part) be due to differences in the formation of DNA-reactive metabolites. Results show that qualitatively, adduct profiles in CAR-R and CAR-S epidermis are similar. Quantitatively, the total binding level is slightly higher in CAR-S versus CAR-R mice during the 30-day follow-up. However, these minor differences do not increase in function of the response to selection observed through three consecutive generations. A 2- or 4-week promotion with 12-O-tetradecanoylphorbol-13-acetate enhances the decrease of adduct level in the two lines. This effect is somewhat more pronounced in CAR-S mice. Results strongly suggest that the expression of the genes responsible for CAR-R/CAR-S phenotypic difference affects mainly the postinitiation stages.

32P-postlabeling analysis of inhibition by norharman of formation of dibenzo[a,e]fluoranthene--DNA adducts in mouse embryo fibroblasts.

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.

Differences in metabolic activation of dibenzo[a,e]fluoranthene characterized by 32P-postlabeling in two mouse fibroblast models.

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.

Non-random distribution of dibenzo[a,e]fluoranthene-induced DNA adducts in DNA loops in mouse fibroblast nuclei.

The three-dimensional distribution of nuclear DNA damage induced by dibenzo(a,e)fluoranthene (DBF), a potent carcinogen for mouse fibroblasts, has been examined. The intact supercoiled nuclear DNA obtained from nucleoids of mouse fibroblasts incubated with DBF was fractionated into loop DNA attached to the matrix (10%) and bulk loop DNA (90%). Preferential binding of DBF to the DNA of the extremities of loops, which are rich in regulatory sequences, was observed in all experiments. An increase of the preferential DBF binding was seen when fibroblasts were incubated with both DBF and novobiocin or hydroxyurea. The excess damage seen in loop DNA attached to the cage may be due to the kinetics of diffusion to the interior of the nucleus of hydrophobic DBF metabolites accumulated in lipid-rich nuclear membrane.

Formation and persistence of DNA-protein cross-links induced in mouse fibroblasts by dibenzo[a,e]fluoranthene, and modulation by stimulators and inhibitors of polycyclic aromatic hydrocarbons (PAH) metabolism.

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.

Formation and removal of dibenzo[a,e]fluoranthene-DNA adducts in mouse embryo fibroblasts.

In vivo binding of dibenzo[a,e]fluoranthene (DBF) to mouse embryo fibroblast DNA was compared with that observed previously in vitro on calf thymus DNA incubated with mouse liver microsomes. The h.p.l.c. elution patterns of the adducts formed by DBF metabolites with DNA and obtained in vivo at the optimal exposure time of 42-48 h were qualitatively very similar to the patterns obtained in vitro, but their amplitude was quantitatively reduced. There are two striking differences between the in vivo and in vitro results. Firstly, the most polar peak A, very abundant in vitro, was absent in vivo. Secondly, the reactivity of the two major proximate metabolites of DBF, the bay and pseudo-bay region dihydrodiols, was very different in intact cells compared with the results in vitro. When incubated in vitro, pseudo-bay region dihydrodiol DBF was twice as reactive as bay region dihydrodiol DBF. The opposite reactivities were observed in vivo. The major DBF-DNA adducts formed in vivo were collected in the peaks E, B and C. The predominant peak E contained DNA adducts of both bay and pseudo-bay region dihydrodiolepoxides which are the major ultimate metabolites of DBF in vivo and in vitro. The other two prominent peaks B and C contained DNA adducts of 3-hydroxy DBF pseudo-bay region dihydrodiolepoxide and the 7-hydroxy DBF bay region dihydrodiolepoxide, respectively. After adduct formation, post incubation of fibroblasts for a further 48 h, in the absence of DBF, eliminated half the amount of adducts present. Peak B adducts were repaired more efficiently than those of peaks E, C D and F. The carcinogenic initiating activity of DBF appears to be a complex process in which several DNA adducts play a role.

DNA-protein crosslinks induced by exposure of cultured mouse fibroblasts to dibenzo[a,e]fluoranthene and its bay- and pseudo-bay region dihydrodiols.

The presence of DNA-protein crosslinks was shown by the alkaline elution technique in cultured mouse fibroblasts treated with dibenzo[a,e]fluoranthene (DBF), a non-alternating carcinogenic PAH. The crosslinks appeared to be between DNA and protein, since the effect disappeared with proteinase treatment. The crosslinking effect increased with time of exposure to DBF. Two major metabolites of DBF were tested under similar conditions. The pseudo-bay region dihydrodiol of DBF induced similar effects. Its isomer, the bay-region dihydrodiol, was inactive. The possible intervention of a bifunctional metabolite of DBF in the DNA-protein crosslinking process is discussed.