Induction of DNA synthesis in mouse liver following increases of DNA adduct levels elicited by very low cumulative doses of the genotoxic hepatocarcinogen 7H-dibenzo[c,g]carbazole.

The purpose of this work was to investigate the administration of very low but repeated doses of a genotoxic carcinogen and an eventual correlation with cellular DNA synthesis. The compound 7H-dibenzo[c,g]carbazole is a genotoxic carcinogen in the mouse liver and was administered topically at the dose of 13.35 microg per animal every 2 days to give a total of 13 applications. Animals were sacrificed 48 hours after every 2 applications until the 10th treatment, then 48 hours after every treatment. Postulated genotoxic effects such as DNA adduct formation were detected by the 32P-post labeling assay. Liver sections were examined for microscopic changes and DNA synthesis. Results showed an increase of the total DNA adduct level in the liver throughout the study with a slowing down in the level after the sixth application of the compound. This change could correspond to the onset of DNA synthesis and to the moderate hepatocellular apoptosis which was observed. The DNA synthesis, which was considered to be secondary to the cytotoxicity induced by the high level of DNA adducts altering normal cellular activity, could also be the opportunity to fix the DNA adducts into heritable mutations. These results raise the question regarding the risk assessment in humans exposed regularly to very low doses of chemicals in the environment: for non-proliferating tissue, the regular accumulation of DNA adducts could remain silent until a "threshold level" is reached from which stimulation of the DNA synthesis may fix the DNA adducts into heritable mutations, eventually leading to tumors.

Tissue-specific activities of methylated dibenzo[c,g]carbazoles in mice: carcinogenicity, DNA adduct formation, and CYP1A induction in liver and skin.

The potent multitissue carcinogen 7H-dibenzo[c,g]carbazole and nine methylated derivatives, synthesized on the basis of the positions in the parent compound that are involved in metabolism, were tested for their ability to induce sarcomas and hepatic tumors in XVIInc/Z mice. In addition, the capacity of these compounds to induce DNA adducts in skin and liver was investigated by (32)P-postlabeling analysis after their topical administration. Induction by these compounds of cytochromes P450 of the 1A family in liver and skin was investigated and correlated to their carcinogenic potential. A clear correlation was found between the tissue specificity of DNA binding and the capacity of each compound to induce skin or liver tumors. In contrast, no direct relationship was observed between the capacity of the compounds to induce cytochromes 1A1/1A2 and the tissue specificity of carcinogenesis or DNA binding in liver or skin. The results are discussed with respect to the positions of methyl groups in the 7H-dibenzo[c,g]carbazole molecule.

Kinetics of DNA adduct formation and removal in mouse hepatocytes following in vivo exposure to 5,9-dimethyldibenzo[c,g]carbazole.

5,9-Dimethyldibenzo[c,g]carbazole (DMDBC), a potent mouse hepatocarcinogen, has been shown to induce a non-linear increase in mutant frequency in the liver of the transgenic MutaMouse. To gain insight into the mechanisms underlying the mutagenicity of DMDBC in vivo, DNA damage formation and removal were monitored in mouse hepatocytes over 4-144 h after a single skin application of 10 or 90 mg/kg DMDBC. DNA adducts were measured by (32)P-post-labeling. DNA repair was assessed by: (i) the unscheduled DNA synthesis (UDS) assay, which measures [(3)H]thymidine incorporation into hepatocyte DNA undergoing excision repair; (ii) the Comet assay, which detects DNA strand breaks transiently produced between the incision and rejoining steps of the excision repair process. A plateau of approximately 400 DNA adducts/10(8) nucleotides was reached 24 h after treatment with 10 mg/kg and remained unchanged until 144 h. UDS activity was significantly induced at 15 and 24 h, while no DNA strand breaks were observed at any sampling time. These results suggest that DNA repair mechanisms were efficiently induced and the formation of a high degree of DNA damage was avoided at this dose level. Following exposure to 90 mg/kg DMDBC, the number of DNA adducts increased sharply to a maximum at 24 h ( approximately 8000/10(8) nucleotides) and then declined to approximately 500/10(8) nucleotides at 144 h. UDS activity was markedly induced from 15 to 72 h. Low levels of DNA strand breaks were observed at 24 and 48 h. The formation of large numbers of DNA adducts and the emergence of DNA strand breaks despite a strong initial induction of UDS activity suggested that DNA repair mechanisms were saturated at this dose level. This phenomenon could partly account for the non-linear induction of gene mutations previously reported in the liver of the transgenic MutaMouse.

Kinetics of induction of DNA adducts, cell proliferation and gene mutations in the liver of MutaMice treated with 5,9-dimethyldibenzo[c,g]carbazole.

5,9-Dimethyldibenzo[c,g]carbazole (DMDBC) is a synthetic derivative of the environmental pollutant 7H-dibenzo[c,g]carbazole. DMDBC is a potent genotoxic carcinogen specific for mouse liver. Using the MutaMouse lacZ transgenic mouse model and a positive selection assay, we measured lacZ mutant frequency (MF) in the liver 28 days after a single s.c. administration of DMDBC at 3, 10, 30, 90 or 180 mg/kg. MF remained low at 3 and 10 mg/kg, but increased markedly from 30 mg/kg onwards. To investigate the reason for this non-linear response, we examined mechanisms potentially involved in mutation induction in the liver. Genotoxic effects such as DNA adduct formation were detected in 32P-post-labelling studies. Liver sections were examined for microscopic changes and cell proliferation. These parameters, and MF, were studied 2, 4, 7, 14, 21 and 28 days after a single s.c. administration of 10 or 90 mg/kg DMDBC. At 10 mg/kg, a dose found to double the MF on day 28, DNA adducts reached a level of 200-600 adducts per 10(8) nucleotides from day 4 to day 28. No changes in histology or cell proliferation were detected at this low dose. At 90 mg/kg, MF increased gradually from day 7 to day 28 (maximum 44-fold). The DNA adduct level ranged from 400 to 4500 adducts per 10(8) nucleotides on day 2, then stabilized at approximately 400 adducts per 10(8) nucleotides on day 4. An early cytotoxic effect was detected microscopically in centrilobular hepatocytes, and was followed by liver cell proliferation. These data suggest that the marked increase in MF in MutaMouse liver after treatment in vivo with DMDBC at 90 mg/kg may be explained by the induction of replicative DNA synthesis due to a cytotoxic effect, allowing the fixation of persistent DNA adducts into mutations.

Inversion of genetic predisposition to carcinogenesis in liver of two lines of mice selected for resistance (Car-R) or susceptibility (Car-S) to skin carcinogenesis.

Two lines of mice, one resistant (Car-R) and one susceptible (Car-S) to skin carcinogenesis, were produced by bi-directional selective breeding. To see whether the characteristics of susceptibility or resistance to tumorigenesis were also expressed in the liver and lung, the two lines were submitted comparatively to treatment with 5,9-dimethyl dibenzo[c,g]carbazole (DiMeDBC), a potent hepatocarcinogenic derivative of the ubiquitous heterocyclic carcinogenic pollutant, 7H-dibenzo[c,g]-carbazole (DBC). An inversion of genetic predisposition to carcinogenesis in liver was observed. Car-R animals displayed rapid tumorigenesis in 100% of cases while Car-S mice were remarkably less sensitive, showing a 4-fold lower mean tumor multiplicity and a 4-month longer latency time. In parallel adduct formation by DiMeDBC and DBC in liver DNA was analyzed by the 32P-postlabeling method, showing a remarkably higher level in Car-R mice than in Car-S animals. These data indicate that tissue-specific sensibility in carcinogenesis may involve gene expression at various levels.

Inhibition of 5,9-dimethyldibenzo[c,g]carbazole-DNA adduct formation in mouse liver by pretreatment with cytochrome P4501A inducers in vivo.

Mice of the XVIInc/Z and DBA/2N strains, which are responsive and nonresponsive, respectively, to the aryl hydrocarbon (Ah) receptor, were treated with the hepatocarcinogen 5,9-dimethyldibenzo[c,g]carbazole and their livers were examined by nuclease P1-enhanced 32P-postlabeling for the levels of DNA adducts formed. Pretreatment at the doses usually reported in the literature with the cytochrome P4501A (CYP1A) inducers 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), beta-naphthoflavone (BNF), and isosafrole modulated DNA adduction. In XVIInc/Z mice, DNA adduction was totally inhibited by TCDD (a CYP1A1/1A2 inducer), BNF (a CYP1A1/1A2 inducer), and isosafrole (a CYP1A2 inducer). In DBA/2N mice, in which DNA adduction was also inhibited by TCDD, about 25% of the DNA adduct levels persisted after pretreatment with BNF (not a CYP1A1/1A2 inducer in this strain) or isosafrole (a CYP1A2 inducer in this strain). The increase (in all cases less than twofold) in the levels of the phase-II drug-metabolizing enzymes glutathione S-transferase and uridine diphospho-glucuronyltransferase after treatment with inducers cannot explain the total disappearance of DNA adducts. Assays of 5-bromo-2'-deoxyuridine incorporation did not show any induction of DNA synthesis which could explain the decrease in adducts. These results suggest that in vivo 1) increases in CYP1A enzymes by inducers are not correlated with enhanced levels of certain DNA adducts; and 2) phase-II drug metabolizing enzymes are not the main cellular protection pathway for detoxification. An additional mechanism, perhaps also induced by the Ah receptor but highly dependent on the dose of inducer, could be involved in parallel to multidrug resistance (mdr); further experiments are needed to identify this process used by the cell to enhance its protection against toxic or genotoxic effects.

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.

DNA adduct formation in primary mouse embryo cells induced by 7H-dibenzo[c,g]carbazole and its organ-specific carcinogenic derivatives.

The nuclease P1 modification of the 32P-postlabeling technique was used to study the biological activity of 7H-dibenzo[c,g]carbazole (DBC) and some of its derivatives, including N-methyldibenzo[c,g]carbazole (N-MeDBC), 5,9-dimethyldibenzo[c,g]carbazole (5,9-diMeDBC), 5,9,N-trimethyldibenzo[c,g]carbazole (5,9,N-triMeDBC), 6-methoxydibenzo[c,g]carbazole (6-McODBC), N-acetyldibenzo[c,g]carbazole (N-AcDBC), N-hydroxymethyldibenzo[c,g]carbazole (N-HMeDBC) in primary mouse embryo cells. A very good correlation was found between carcinogenic specificity in vivo of these N-heterocyclic aromatic hydrocarbons and their DNA-adduction in vitro. Primary mouse embryo cells were able to metabolize and detect tissue-specific sarcomagens N-MeDBC and 6-MeODBC as well as derivatives with both sarcomagenic and hepatocarcinogenic activity, DBC, N-AcDBC, and N-HMeDBC. The strong specific hepatocarcinogen 5,9-diMeDBC in vivo, did not induce any DNA-adducts in the embryo cells, which suggests that the enzymatic composition of the target tissue probably is the determining factor in the organ specificity of this derivative. 5,9,N-triMeDBC, derivative without any carcinogenic activity in vivo, did not induce any DNA-adducts in primary mouse embryo cells. Pretreatment of cells with 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) apparently stimulated DNA-adduct formation in the cells exposed to DBC, 6-MeODBC, and N-MeDBC. No or a very slight effect of TCDD on DNA-adduct formation was found in cells exposed to N-HMeDBC and N-AcDBC. Preliminary results have shown that TCDD slightly induced cytochrome P4501A1-linked ethoxyresorufin O-deethylase (EROD) activity in primary mouse embryo cells. These data suggest the role of cytochrome P4501A1 in the metabolism of DBC derivatives with sarcomagenic activity.

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.

Characterization and evaluation by 32P-postlabelling of psoralen-type DNA adducts in HeLa cells.

Samples of DNA irradiated at 405 and/or 365 nm in the presence of 8-methoxypsoralen (8-MOP) were analysed via a modified postlabelling assay using three hydrolysis enzymes other than those employed previously. These enzymes (deoxyribonucleaseI, venom phosphodiesterase and alkaline phosphatase) liberated 3'-adducted dinucleotide monophosphate instead of the 5'-modified dinucleotide monophosphate normally obtained. The first separation chromatography (D1) of samples irradiated in the presence of 8-MOP showed a single spot above the origin, and the next separation (D2) resolved this spot into two components (spots I and II). Double irradiation experiments in which samples of DNA were first irradiated at 405 nm before being irradiated at 365 nm showed that spot II could be transformed into spot I. The use of 6,4,4'-trimethylangelicin, which induced only photomonoadducts under UVA irradiation, gave only spot II. These two results indicated that spots I and II were respectively due to interstrand cross-links and monoadducts. Dose-effect experiments showed that spots I and II were dose dependent, and low-dose irradiations permitted us to measure one interstrand cross-link and two monoadducts per 10(8) base pairs.

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.

32P-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 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.

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.

Marked differences between mutagenicity in Salmonella and tumour-initiating activities of dibenzo[a,e]fluoranthene proximate metabolites; initiation inhibiting activity of norharman.

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.

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.

Possible involvement of a vicinal, non-bay-region dihydrodiol-epoxide in the activation of dibenzo[a,e]fluoranthene into bacterial mutagens.

Dibenzo[a,e]fluoranthene, its 7-hydroxy, 3,4- and 12,13-dihydrodiol metabolic derivatives as well as three synthetic, structurally related hydrocarbons, were tested for mutagenicity towards Salmonella typhimurium TA100 strain in the presence of 3-methylcholanthrene-treated rat and mouse liver post-mitochondrial supernatants. Of these compounds, the 12,13-dihydrodiol showed the highest activity, being 6-10 times more mutagenic than the parent compound. Our data, in conjunction with those of previous studies on the liver microsomal metabolism and DNA binding of dibenzo[a,e]fluoranthene and its dihydrodiols, indicate that activation of dibenzo[a,e]fluoranthene to bacterial mutagens may occur predominantly through a vicinal, non-bay-region 12,13-dihydrodiol epoxide.

The metabolic activation of dibenzo[a,e]fluoranthene in vitro. Evidence that its bay-region and pseudo-bay-region diol-epoxides react preferentially with guanosine.

Dibenzo[a,e]fluoranthene ( DBF ), a non- alternant carcinogenic polycyclic aromatic hydrocarbon (PAH), binds covalently to DNA. The main adducts were characterized as covalent additions of its bay-region and pseudo-bay-region diol-epoxides. The structure of these 2 adducts was analyzed by mass spectrometry using their persilyl derivatives. 3,4-Dihydroxy-1,2-epoxy-1,2,3,4-tetrahydro- DBF (3,4-diol-1,2-epoxy- DBF ) and 12,13-dihydroxy-10,11-epoxy-10,11,12,13-tetrahydro- DBF (12, 13-diol-10,11-epoxy- DBF ) obtained by synthesis were allowed to react in vitro with calf thymus DNA or with poly(G). The comigration of DNA and poly(G) adducts isolated after acid hydrolysis of DNA and poly(G) was in good agreement with mass spectroscopic results: both bay-region and pseudo-bay-region DBF diol-epoxides reacted with guanine residues.

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.