Comparison of cytochrome P450- and peroxidase-dependent metabolic activation of the potent carcinogen dibenzo[a,l]pyrene in human cell lines: formation of stable DNA adducts and absence of a detectable increase in apurinic sites

The potent carcinogen dibenzo[a,l]pyrene (DB[a,l]P) has been reported to form both stable and depurinating DNA adducts upon activation by cytochrome P450 enzymes and/or cellular peroxidases. Only stable DB[a,l]P-DNA adducts were detected in DNA after reaction of DB[a,I]P-11,12-diol-13,14-epoxides in solution or cells in culture. To determine whether DB[a,l]P can be activated to metabolites that form depurinating adducts in cells with either high peroxidase (human leukemia HL-60 cell line) or cytochrome P450 activity (human mammary carcinoma MCF-7 cell line), cultures were treated with DB[a,l]P for 4 h, and the levels of stable adducts and apurinic (AP) sites in the DNA were determined. DNA samples from DB[a,l]P-treated HL-60 cells contained no detectable levels of either stable adducts or AP sites. MCF-7 cells exposed to 2 microM DB[a,l]P for 4 h contained 4 stable adducts per 10(6) nucleotides, but no detectable increase in AP sites. The results indicate that metabolic activation of DB[a,l]P by cytochrome P450 enzymes to diol epoxides that form stable DNA adducts, rather than one-electron oxidation catalyzed either by cytochrome P450 enzymes or peroxidases to form AP sites, is responsible for the high carcinogenic activity of DB[a,l]P.     

Effect of a complex environmental mixture from coal tar containing polycyclic aromatic hydrocarbons (PAH) on the tumor initiation, PAH-DNA binding and metabolic activation of carcinogenic PAH in mouse epidermis.

Human exposure to polycyclic aromatic hydrocarbons (PAH) occurs through complex mixtures such as coal tar. The effect of complex PAH mixtures on the activation of carcinogenic PAH to DNA-binding derivatives and carcinogenesis were investigated in mice treated topically with NIST (National Institute of Standards and Technology) Standard Reference Material 1597 (SRM), a complex mixture of PAH extracted from coal tar, and either additional benzo[a]pyrene (B[a]P) or dibenzo[a,l]pyrene (DB[a,l]P). In an initiation-promotion study using 12-O-tetradecanoylphorbol-13-acetate as the promoter for 25 weeks, the SRM and B[a]P co-treated mice had a similar incidence of papillomas per mouse compared with the group exposed to B[a]P alone as the initiator. PAH-DNA adduct analysis of epidermal DNA by 33P-post-labeling and reversed-phase high-performance liquid chromatography found the SRM co-treatment led to a significant decrease in the total level of DNA adducts and B[a]P-DNA adducts to less than that observed in mice treated with B[a]P alone at 6, 12 and 72 h exposure. After 24 and 48 h exposure, there was no significant difference in the levels of adducts between these groups. In the DB[a,l]P initiation-promotion study, the co-treated group had significantly fewer papillomas per mouse than mice treated with DB[a,l]P alone as initiator. Averaging over the times of exposure gave strong evidence that mice co-treated with SRM and DB[a,l]P had a significantly lower level of PAH-DNA adducts than mice treated with DB[a,l]P alone. Western immunoblots showed that both cytochrome P450 (CYP) 1A1 and 1B1 were induced by the SRM. These results are consistent with the hypothesis that two major factors determining the carcinogenic activity of PAH within a complex mixture are (i) the persistence of certain PAH-DNA adducts as well as total adduct levels, and (ii) the ability of the components present in the mixture to inhibit the activation of carcinogenic PAH by the induced CYP enzymes.     

Comparison of the morphological transforming activities of dibenzo[a,l]pyrene and benzo[a]pyrene in C3H10T1/2CL8 cells and characterization of the dibenzo[a,l]pyrene-DNA adducts

C3H10T1/2CL8 (C3H10T1/2) mouse embryo fibroblasts were used to study the in vitro carcinogenic activities of dibenzo[a,l]pyrene (DB[a,l]P) and benzo[a]pyrene (B[a]P). The morphological transforming activities of these rodent carcinogens were compared using replicate concentration- response studies. In concentration ranges where both polycyclic aromatic hydrocarbons (PAHs) were active, DB[a,l]P proved to be four to 12 times as potent as B[a]P based on concentration. At lower concentrations DB[a,l]P was active at 0.10 and 0.20 microM, concentrations where B[a]P was inactive. This makes DB[a,l]P the most potent non-methylated PAH evaluated to date in C3H10T1/2 cells. DNA adducts of DB[a,l]P in C3H10T1/2 cells were analyzed by both TLC and TLC/HPLC 32P-postlabeling methods using mononucleotide 3'-phosphate adduct standards derived from the reactions of anti-DB[a,l]P-11,12-diol- 13,14-epoxide (anti-DB[a,l]PDE) and syn-DB[a,l]P-11,12-diol-13,14- epoxide (syn-DB[a,l]PDE) with deoxyadenosine 3'-monophosphate and deoxyguanosine 3'-monophosphate. All of the DNA adducts observed in C3H10T1/2 cells treated with DB[a,l]P were identified as being derived from the metabolism of DB[a,l]P to its fjord region diol epoxides through DB[a,l]P-11,12-diol. The predominant adduct was identified as an anti-DB[a,l]PDE-deoxyadenosine adduct. Other major adducts were anti- DB[a,l]PDE-deoxyguanosine and syn-DB[a,l]PDE-deoxyadenosine adducts with minor amounts of syn-DB[a,l]PDE-deoxyguanosine adducts. These DNA adduct data are consistent with similar findings of DB[a,l]PDE- deoxyadenosine adducts in mouse skin studies and human mammary cells in culture.      

Modulation of gene expression and DNA adduct formation in HepG2 cells by polycyclic aromatic hydrocarbons with different carcinogenic potencies

Polycyclic aromatic hydrocarbons (PAHs) can occur in relatively high concentrations in the air, and many PAHs are known or suspected carcinogens. In order to better understand differences in carcinogenic potency between PAHs, we investigated modulation of gene expression in human HepG2 cells after 6 h incubation with varying doses of benzo[a]pyrene (B[a]P), benzo[b]fluoranthene (B[b]F), fluoranthene (FA), dibenzo[a,h]anthracene (DB[a,h]A), 1-methylphenanthrene (1-MPA) or dibenzo[a,l]pyrene (DB[a,l]P), by using cDNA microarrays containing 600 toxicologically relevant genes. Furthermore, DNA adduct levels induced by the compounds were assessed with (32)P-post-labeling, and carcinogenic potency was determined by literature study. All tested PAHs, except 1-MPA, induced gene expression changes in HepG2 cells, although generally no dose-response relationship could be detected. Clustering and principal component analysis showed that gene expression changes were compound specific, since for each compound all concentrations grouped together. Furthermore, it showed that the six PAHs can be divided into three groups, first FA and 1-MPA, second B[a]P, B[b]F and DB[a,h]A, and third DB[a,l]P. This grouping corresponds with the carcinogenic potencies of the individual compounds. Many of the modulated genes are involved in biological pathways like apoptosis, cholesterol biosynthesis and fatty acid synthesis. The order of DNA adduct levels induced by the PAHs was: B[a]P > DB[a,l]P > B[b]F > DB[a,h]A > 1-MPA >/= FA. When comparing the expression change of individual genes with DNA adduct levels, carcinogenic potency or Ah-receptor antagonicity (the last two were taken from literature), several highly correlated genes were found, of which CYP1A1, PRKCA, SLC22A3, NFKB1A, CYP1A2 and CYP2D6 correlated with all parameters. Our data indicate that discrimination of high and low carcinogenic PAHs by gene expression profiling is feasible. Also, the carcinogenic PAHs induce several pathways that were not affected by the least carcinogenic PAHs.     

Comparative dose-response tumorigenicity studies of dibenzo[alpha,l]pyrene versus 7,12-dimethylbenz[alpha]anthracene, benzo[alpha]pyrene and two dibenzo[alpha,l]pyrene dihydrodiols in mouse skin and rat mammary gland

Comparative studies were conducted of the tumor-initiating activity in mouse skin and carcinogenicity in rat mammary gland of dibenzo[a,l]pyrene (DB[a,l]P) versus 7,12-dimethyl-benz[a]anthracene (DMBA), the most potent recognized carcinogenic polycyclic aromatic hydrocarbon (PAH); benzo[a]pyrene (B[a]P), the most potent recognized carcinogenic environmental PAH; DB[a,l]P 8,9-dihydrodiol, the K-region dihydrodiol; and DB[a,l]P 11,12-dihydrodiol, precursor to the bay-region diolepoxide. The tumor-initiating activity of DB[a,l]P and B[a]P was compared in the skin of female SENCAR mice at doses of 300, 100 and 33.3 nmol. The mice were promoted with 12-O-tetradecanoylphorbol-13-acetate (TPA) twice-weekly for 13 weeks. DB[a,l]P at all doses induced significantly more tumors than B[a]P at the corresponding dose, with a significantly shorter latency. Subsequently, the tumor-initiating activity of DB[a,l]P was compared in the skin of female SENCAR mice to that of DMBA, B[a]P, DB[a,l]P 8,9-dihydrodiol and DB[a,l]P 11,12-dihydrodiol at doses of 100, 20 and 4 nmol. The mice were promoted with TPA twice-weekly for 24 weeks. In addition, groups of mice were initiated with 100 nmol of DB[a,l]P, DMBA, B[a]P, DB[a,l]P 8,9-dihydrodiol or DB[a,l]P 11,12-dihydrodiol and kept without promotion. This experiment showed that in the mouse skin, DB[a,l]P and DB[a,l]P 11,12-dihydrodiol displayed similar tumor-initiating activity with a response inversely proportional to the dose, presumably due to the toxicity of the compounds. At the high dose they elicited tumors earlier than DMBA, though DMBA produced a much higher tumor multiplicity. At the low dose, DMBA, DB[a,l]P and DB[a,l]P 11,12-dihydrodiol exhibited similar tumorigenicities. DB[a,l]P 8,9-dihydrodiol was a marginal tumor initiator. Once again, DB[a,l]P was by far a much stronger tumor initiator than B[a]P. Female Sprague-Dawley rats were treated with 1.0 or 0.25 mumol of DB[a,l]P, DMBA or B[a]P by intramammillary injection at eight teats. DB[a,l]P at both doses was a more potent carcinogen than DMBA at the corresponding dose in the rat mammary gland. B[a]P was a marginal mammary carcinogen, eliciting only a few fibrosarcomas. Thus, these data suggest that DB[a,l]P is the strongest PAH carcinogen ever tested.     

Inhibition of the binding of 7,12-dimethylbenz[a]anthracene and benzo[a]pyrene to DNA in mouse skin epidermis by 1-ethynylpyrene

The effects of 1-ethynylpyrene (EP), 1-vinylpyrene (VP) and 2-ethynlnaphthalene (EN) on the covalent binding of 7,12-dimethylbenz[a]anthracene (DMBA) and of benzo[a]-pyrene (B[a]P) to the epidermal DNA in mouse skin were investigated. When applied topically, 5 min before an initiating dose of 10 nmol DMBA or of 200 nmol B[a]P, EP was an effective inhibitor of the formation of the covalent complexes of these procarcinogenic polycyclic aromatic hydrocarbons (PAHs) with the epidermal DNA. VP, applied under the same conditions, was a significantly less effective inhibitor of the binding of DMBA to DNA and showed even weaker inhibition of the binding of B[a]P. EN was ineffective as an inhibitor of the binding of either DMBA or B[a]P. These results establish that both the pyrene nucleus and the ethynyl substituent of EP contribute to the effective inhibition of the binding of DMBA and B[a]P to the epidermal DNA of mouse skin. No significant changes in the ratios of the anti- to the syndiol epoxide-DNA adducts of DMBA or of B[a]P were produced by doses of EP that produced inhibitions of the binding to DNA. At doses of VP that inhibited covalent binding of both DMBA and B[a]P, no changes in DMBA-DNA adduct distributions were observed but changes in the relative proportions of several B[a]P-DNA adducts were noted. These data are discussed in terms of the potential of aryl acetylenes to act as suicide inhibitors (mechanism-based inactivators) of cytochrome P450-dependent monooxygenase isozymes.     

Dibenzo[a,l]pyrene-induced DNA adduction, tumorigenicity, and Ki-ras oncogene mutations in strain A/J mouse lung

Dibenzo[a,l]pyrene (DB[a,l]P), an environmental polycyclic aromatic hydrocarbon, is the most potent carcinogen ever tested in mouse skin and rat mammary gland. In this study, DB[a,l]P was examined for DNA adduction, tumorigenicity, and induction of Ki-ras oncogene mutations in tumor DNA in strain A/J mouse lung. Groups of mice received a single i.p. injection of 0.3, 1.5, 3.0, or 6.0 mg/kg DB[a,l]P in tricaprylin. Following treatment, DNA adducts were measured at times between 1 and 28 days, while tumors were counted at 250 days and analyzed for the occurrence of point mutations in codons 12 and 61 of the Ki-ras oncogene. DB[a,l]P in strain A/J mouse lung induced six major and four minor DNA adducts. Maximal levels of adduction occurred between 5 and 10 days after injection followed by a gradual decrease. DB[a,l]P-DNA adducts in lung tissue were derived from both anti- and syn-11,12- dihydroxy-13,14-epoxy- 11,12,13,14-tetrahydrodibenzo[a,l]pyrene (DB[a,l]PDE) and both deoxyadenosine (dAdo) and deoxyguanosine (dGuo) residues in DNA as revealed by cochromatography. The major adduct was identified as a product of the reaction of an anti-DB[a,l]PDE with dAdo in DNA. DB[a,l]P induced significant numbers of lung adenomas in a dose- dependent manner, with the highest dose (6.0 mg/kg) yielding 16.1 adenomas/mouse. In tricaprylin-treated control animals, there were 0.67 adenomas/mouse. Based on the administered dose, DB[a,l]P was more active than other environmental carcinogens including benzo[a]pyrene. As a function of time-integrated DNA adduct levels, DB[a,l]P induced lung adenomas with about the same potency as other PAHs, suggesting that the adducts formed by DB[a,l]P are similar in carcinogenic potency to other PAHs in the strain A/J mouse lung model. Analysis of the Ki- ras mutation spectrum in DB[a,l]P-induced lung tumors revealed the predominant mutations to be G-->T transversions in the first base of codon 12, A-->G transitions in the second base of codon 12, and A-->T transversions in the second or third base of codon 61, concordant with the DNA adduct profile.      

Binary PAH mixtures cause additive or antagonistic effects on gene expression but synergistic effects on DNA adduct formation

Polycyclic aromatic hydrocarbons (PAHs) cover a wide range of structurally related compounds which differ greatly in their carcinogenic potency. PAH exposure usually occurs through mixtures rather than individual compounds. Therefore, we assessed whether the effects of binary PAH mixtures on gene expression, DNA adduct formation, apoptosis and cell cycle are additive compared with the effects of the individual compounds in human hepatoma cells (HepG2). Equimolar and equitoxic mixtures of benzo[a]pyrene (B[a]P) with either dibenzo[a,l]pyrene (DB[a,l]P), dibenzo[a,h]anthracene (DB[a,h]A), benzo[b]fluoranthene (B[b]F), fluoranthene (FA) or 1-methylphenanthrene (1-MPA) were studied. DB[a,l]P, B[a]P, DB[a,h]A and B[b]F dose-dependently increased apoptosis and blocked cells cycle in S-phase. PAH mixtures showed an additive effect on apoptosis and on cell cycle blockage. DNA adduct formation in mixtures was higher than expected based on the individual compounds, indicating a synergistic effect of PAH mixtures. Equimolar mixtures of B[a]P and DB[a,l]P (0.1, 0.3 and 1.0 microM) were assessed for their effects on gene expression. Only at 1.0 microM, the mixture showed antagonism. All five compounds were also tested as a binary mixture with B[a]P in equitoxic concentrations. The combinations of B[a]P with B[b]F, DB[a,h]A or FA showed additivity, whereas B[a]P with DB[a,l]P or 1-MPA showed antagonism. Many individual genes showed additivity in mixtures, but some genes showed mostly antagonism or synergism. Our results show that the effects of binary mixtures of PAHs on gene expression are generally additive or slightly antagonistic, suggesting no effect or decreased carcinogenic potency, whereas the effects on DNA adduct formation show synergism, which rather indicates increased carcinogenic potency.     

The K-region trans-8,9-diol does not significantly contribute as an intermediate in the metabolic activation of dibenzo[a,l]pyrene to DNA-binding metabolites by human cytochrome P450 1A1 or 1B1.

Metabolic activation of the K-region trans-8,9-diol of the highly carcinogenic hexacyclic aromatic hydrocarbon dibenzo[a,l]pyrene (DB[a,l]P) by human cytochrome P-450 (P450) 1A1 and 1B1 was investigated in Chinese hamster V79 cell lines expressing human P450 1A1 or 1B1. P450 1A1 and 1B1 are the major P450s involved in metabolic activation of polycyclic aromatic hydrocarbons in human cells. The major DNA adducts formed by metabolism of DB[a,l]P in cultures expressing P450 1A1 or 1B1 resulted mainly from the fjord region (-)-anti-DB[a,l]P-11,12-diol 13,14-epoxide [(-)-anti-DB[a,l]PDE] and, to a lesser extent, (+)-syn-DB[a,l]PDE. In V79 cells expressing human P450 1A1, high amounts of as yet unidentified highly polar DNA adducts are formed in addition to the DNA adducts derived from DB[a,l]PDEs. Human P450 1A1 has been found to metabolize DB[a,l]P on its K-region to the trans-8,9-diol, and it has been proposed that the DNA binding of the parent compound in P450 1A1-expressing tissues may be partially mediated by activation of the K-region trans-8,9-diol to form bis-diol epoxides. V79 cells expressing human P450 1A1 or 1B1 formed only low amounts of DNA adducts after treatment with high doses of the K-region trans-8,9-diol. None of the adducts formed were identical to the main adducts formed in the same cell lines by metabolic activation of DB[a,l]P or (-)-DB[a,l]P-trans-11,12-diol. These results demonstrate that the K-region trans-8,9-diol does not significantly contribute to the genotoxicity of the very potent carcinogen DB[a,l]P in human cells or tissues expressing P450 1A1 or 1B1.     

Dibenzo[A,L]pyrene-induced genotoxic and carcinogenic responses are dramatically suppressed in aryl hydrocarbon receptor-deficient mice

Dibenzo[a,l]pyrene (DB[a,l]P), a notorious air pollutant, is the most powerful carcinogenic polycyclic aromatic hydrocarbon (PAH) ever tested. Although the carcinogenicity of PAH may be primarily mediated by the aryl hydrocarbon receptor (AhR), the in vivo role of AhR in skin carcinogenesis remains to be defined. In this context, we investigated the genotoxic and carcinogenic responses of the AhR-deficient mouse skin to DB[a,l]P. A single painting resulted in a striking epidermal hyperplasia in AhR+/+ mice but not in AhR-/- mice. Bromodeoxyuridine-labeling index and accumulation of p53 protein in epidermal cells of AhR+/+ mice were 8- and 33-fold higher than those of AhR-/- mice, respectively. 32P-Postlabeling assay for DB[a,l]P-DNA adducts displayed a 2-fold increase in the AhR+/+ mouse skin. After DB[a,l]P exposure, AhR-/- mice arranged a nearly 60% reduction in the induction of epidermal cytochrome P450 (CYP)1A1, but CYP1B1 was constitutively expressed in both genotypes of mice, irrespective of DB[a,l]P treatment. As compared with AhR+/+ mice, AhR-/- mice had both significantly lower incidence (100% vs. 33%) and multiplicity (2.7 vs. 0.46) of skin tumors by the complete carcinogenesis study. These observations indicate that a reduced tumor yield in AhR-/- mice may be secondary to reduction of inducible CYP1A1 activation and subsequent DNA adduction. It is evident from our continuous work that although AhR is likely to play a central role in epidermal proliferation and possibly neoplastic transformation, the relative importance of AhR for carcinogenesis may be different among PAH examined.     

8,9-dihydroxy-8,9-dihydrodibenzo[a,l]pyrene is a potent morphological cell-transforming agent in C3H10T(1)/(2)Cl8 mouse embryo fibroblasts in the absence of detectable stable covalent DNA adducts

The comparative genotoxic effects of racemic trans-8,9-dihydroxy-8, 9-dihydrodibenzo[a,l]pyrene (trans-DB[a,l]P-8,9-diol), the metabolic K-region dihydrodiol of dibenzo[a,l] pyrene (DB[a,l]P) (dibenzo[def, p]chrysene) and DB[a,l]P in transformable mouse embryo C3H10T(1)/(2)Cl8 (C3H10T(1)/(2)) fibroblasts was investigated. The C3H10T(1)/(2) mouse embryo morphological cell-transforming activities of these polycyclic aromatic hydrocarbons (PAHs) were assayed using concentration-response studies. At concentrations of 33 nM and above both trans-DB[a,l]P-8,9-diol and DB[a,l]P produced significant (and similar) numbers of type II and III foci per dish and numbers of dishes with type II and II foci. Concomitant cytotoxicity studies revealed a reduction in colony survival of approximately 25% up to 198 nM for both PAHs. DNA adducts of trans-DB[a,l]P-8,9-diol and DB[a,l]P in C3H10T(1)/(2) cells were analyzed by a (32)P-post-labeling TLC/HPLC method. No adducts were observed in the DNA of C3H10T(1)/(2) cells treated with trans-DB[a, l]P-8,9-diol at concentrations that induced morphological cell transformation. Under the same exposure and chromatographic conditions, DNA adducts of deoxyadenosine and deoxyguanosine derived from the fjord region anti-DB[a,l]P-11,12-diol-13,14-epoxide and syn-DB[a,l]P-11,12-diol-13,14-epoxide were observed in the DNA of DB[a,l]P-treated cells. These results indicate that trans-DB[a,l]P-8, 9-diol has intrinsic genotoxic activity equal to that of DB[a,l]P, based on morphological cell transformation of mouse embryo fibroblasts. The activity of trans-DB[a,l]P-8,9-diol is apparently not associated with the formation of observable stable covalent DNA adducts. These results suggest that under appropriate conditions, trans-DB[a,l]P-8,9-diol may serve as an intermediate in the genotoxicity of DB[a,l]P.     

Benzo(e)pyrene-induced alterations in the binding of benzo(a)pyrene and 7,12-dimethylbenz(a)anthracene to DNA in Sencar mouse epidermis

Benzo(e)pyrene [B(e)P] cotreatment slightly increases the tumor-initiating activity of benzo(a)pyrene [B(a)P] and greatly decreases the tumor-initiating activity of 7,12-dimethylbenz(a)anthracene (DMBA) in Sencar mice (DiGiovanni et al., Carcinogenesis 3: 371-375, 1982). The effects of B(e)P on the binding of B(a)P and DMBA to Sencar mouse epidermis were investigated using a protocol similar to the mouse skin tumorigenicity studies. After 12 h of exposure to 50 nmol [3H]B(a)P and low or high doses of B(e)P, the level of [3H]B(a)P bound to mouse epidermal DNA increased by 30%. However, after 24 h exposure to 50 nmol [3H]B(a)P and after 12 or 24 h of exposure to 200 nmol [3H]B(a)P, B(e)P had no effect on the amount of [3H]B(a)P bound to DNA. The ration of anti-(the isomer with the epoxide and benzylic hydroxyl on opposite faces of the molecule) B(a)P-7,8-diol-9,10-epoxide [B(a)PDE]-deoxyribonucleoside adducts to syn- (the isomer with the epoxide and benzylic hydroxyl on the same face of the molecule) B(a)PDE-deoxyribonucleoside adducts did not change at either initiating dose of B(a)P or at any time regardless of the dose of B(e)P. After 12 h of exposure to high doses of B(e)P and a 50-nmol initiating dose of B(a)P the level of [3H]B(a)P bound to DNA increased but there was no change in the proportion of particular B(a)PDE-deoxyribonucleoside adducts present. In contrast, B(e)P inhibited the binding of initiating doses of DMBA (5 and 20 nmol) to DNA after 12 and 48 h of exposure to all dose ratios of B(e)P:DMBA tested. The three major adducts, tentatively identified as anti-DMBA-3,4-diol-1,2-epoxide (DMBADE):deoxyguanosine, syn-DMBADE:deoxyadenosine and anti-DMBADE:deoxyadenosine, decreased to the same relative extent as the dose of B(e)P increased. Thus, the effects of B(e)P on the total binding of these hydrocarbons to DNA in epidermis correlate with the cocarcinogenic and anticarcinogenic effects of B(e)P on B(a)P and DMBA, respectively, in a mouse skin initiation-promotion assay. These results indicate that the mechanism of the co- or anticarcinogenic action of hydrocarbons such as B(e)P involves alteration of the binding of carcinogenic hydrocarbons to DNA. They also suggest that measurement of carcinogenic hydrocarbon-DNA adducts formed during cotreatment with other hydrocarbons will provide a rapid method for predicting the co- or anticarcinogenic effect of the other hydrocarbons.     

Anticarcinogenic and cocarcinogenic effects of benzo[e]pyrene and dibenz[a,c]anthracene on skin tumor initiation by polycyclic hydrocarbons

In the present study, we have examined the effects of benzo[e]pyrene(B[e]P) and dibenz[a,c]anthracene (DB[a,c]A) on the skin tumor-initiatingactivities of methylated and nonmethylated polycyclic aromatichydrocarbons (PAH). B[e]P, when applied 5 min prior to initiationwith seven different PAH skin carcinogens, effectively inhibitedthe tumorinitiating activities of 7,12-dimethylbenz[a]anthracene(DMBA) and dibenz[a,h]anthracene (DB[a,h]A) but had little orno effect on the tumor-initiating activities of 3-methylcholanthrene(MCA), 7-methylbenz[a]anthracene (7-MBA), 12-methylbenz[a]anthracene(12-MBA), and 5-methylchrysene (5-MeC). B[e]P potentiated thetumor-initiating activity of benzo[a]pyrene (B[a]P) by 30%.DB[a,c]A, when applied 5 min prior to initiation, inhibitedthe tumor-initiating activities of DMBA, MCA, and DB[a,h]A buthad little or no effect on the tumor-initiating activities ofB[a]P, 7-MBA, 12-MBA, and 5-MeC. DB[a,c]A, when applied 12,24, or 36 h prior to initiation with B[a]P, which allowed timefor induction of epidermal monooxygenase enzymes, inhibitedtumor initiation. The covalent binding of DMBA and B[a]P toepidermal DNA was examined under the influence of B[e]P. Dosesof 20 and 200 nmol B[e]P given 5 min prior to 10 nmol [³H]DMBAreduced binding to 47 and 22%, respectively, of the controlvalue. In contrast, doses of 200 or 2000 nmol B[e]P given 5min prior to 200 nmol [³H]B[a]P had little or no effect on totalbinding. The data indicate that one cannot predict anti andcocarcinogenic effects of B[e]P and DB[a,c]A on the basis ofa presence or absence of a methyl substituent. In addition,fundamental differences exist in the processing and metabolismof DMBA and B[a]P by mouse epidermal cells.     

DNA damage, repair, and mutation induction by (+)-Syn and (-)-anti-dibenzo[a,l]pyrene-11,12-diol-13,14-epoxides in mouse cells

Polycyclic aromatic hydrocarbons (PAHs) are ubiquitous environmental carcinogens. PAHs are classified into bay and fjord region compounds according to structural differences in the molecule region where enzymatic epoxidation occurs. Dibenzo[a,l]pyrene (DB[a,l]P), one of the fjord region compounds, has been demonstrated to be the most carcinogenic PAH known to date. DB[a,l]P is activated to fjord region (+)-syn and (-)-anti-11,12-dihydroxy-13,14-epoxy-11,12,13,14-tetrahydrodibenzo[a,l]pyrene (DB[a,l]PDE) metabolites. In this study, we analyzed mutagenesis induced by (+)-syn- and (-)-anti-DB[a,l]PDE at the cII transgene in Big-Blue mouse cells. The mutant frequency of untreated cells (background level) was 6.53 x 10(-5). This level increased 3.7-fold for 20 nmol/L, 5.3-fold for 50 nmol/L, and 7.9-fold for 100 nmol/L (+)-syn-DB[a,l]PDE, respectively. In the case of (-)-anti-DB[a,l]PDE it increased 4.5-fold for 20 nmol/L, 6.7-fold for 50 nmol/L, and 10.6-fold for 100 nmol/L, respectively, indicating that (-)-anti-DB[a,l]PDE is slightly more mutagenic than (+)-syn-DB[a,l]PDE. The mutational spectra of (+)-syn- and (-)-anti-DB[a,l]PDE were quite similar except for several hotspots, specific for either (+)-syn-DB[a,l]PDE or (-)-anti-DB[a,l]PDE. The most frequently induced mutations were A to T transversions, which were 43.9% for (+)-syn- and 38.8% for (-)-anti-DB[a,l]PDE. In addition, G to T transversions were induced significantly, at frequencies of 18.5% by (+)-syn- and 18.1% by (-)-anti-DB[a,l]PDE. Using UvrABC cleavage and ligation-mediated PCR or the terminal transferase-dependent PCR method, we have determined DB[a,l]PDE-DNA adduct formation sites and repair rates in carcinogen-exposed cells. The mutation hotspots coincided with sites of strong adduct formation, but not all of the adduct hotspots were mutational hotspots. Slow adduct removal occurred for both (+)-syn- and (-)-anti-DB[a,l]PDE adducts over a time period of up to 72 hours. The data suggest that, although the (-)-anti-isomer is slightly more mutagenic, DNA adducts of both DB[a,l]PDE stereoisomers may have similar biological properties. We discuss the implications of these findings for human cancer mutagenesis.     

Effect of naturally occurring coumarins on the formation of epidermal DNA adducts and skin tumors induced by benzo[a]pyrene and 7,12- dimethylbenz[a]anthracene in SENCAR mice

Several naturally occurring coumarins previously found to be potent inhibitors of mouse hepatic ethoxyresorufin-O-deethylase (EROD) and/or pentoxyresorufin-O-dealkylase (PROD) were examined for their effects on formation of benzo[a]pyrene (B[a]P) and 7,12-dimethylbenz[a]anthracene (DMBA) DNA adducts in mouse epidermis, as well as, their effects on skin tumor initiation by these polycyclic aromatic hydrocarbons (PAH). Bergamottin, a potent inhibitor of hepatic EROD, given topically 5 min prior to an initiating dose of B[a]P, significantly decreased total covalent binding of B[a]P to DNA in a dose-dependent manner 24 h after treatment. A dose of 400 nmol bergamottin reduced covalent binding of B[a]P by 72%. Coriandrin, at a dose of 400 nmol also significantly reduced total covalent binding of B[a]P by 59%. In addition, formation of the major (+)anti-B[a]P-diol epoxide-N2-dGuo adduct was selectively reduced by both of these coumarins. In contrast, bergamottin and coriandrin did not significantly decrease covalent binding of DMBA to epidermal DNA at doses of either 400 nmol or 800 nmol. Imperatorin and isopimpinellin, which are more potent inhibitors of hepatic PROD activity, significantly reduced overall binding of DMBA to epidermal DNA by 67% and 52%, respectively, when applied at doses of 400 nmol. These two coumarins also inhibited B[a]P-DNA adduct formation at similar doses but to a lesser extent. Imperatorin at a dose of 400 nmol dramatically decreased formation of covalent DNA adducts derived from both the anti and syn diol epoxides of DMBA. Bergamottin was a potent inhibitor of tumor initiation by B[a]P while coriandrin was less effective in this regard. Imperatorin was an effective inhibitor of skin tumor initiation by DMBA and also inhibited complete carcinogenesis by this PAH. At dose levels higher than those effective against DMBA, imperatorin also inhibited tumor initiation by B[a]P. The results demonstrate that several naturally occurring coumarins possess the ability to block DNA adduct formation and tumor initiation by PAHs such as B[a]P and DMBA. The mechanism for reduced DNA adduct formation and tumor initiation appears to involve inhibition of the P450s involved in the metabolic activation of these hydrocarbons. Finally, the differential effects of certain coumarins on B[a]P vs DMBA DNA adduct formation and tumor initiation may be useful for dissecting the role of specific cytochromes P450 in their metabolic activation.      

Covalent binding of dibenzpyrenes and benzo[a]Pyrene to DNA: evidence for synergistic and inhibitory interactions when applied in combination to mouse skin

Several well-documented examples of human exposure to car cinogensinvolve complex mixtures of polycydlic aromatic hydrocarbons(PAHs). Although the biological properties of many pure PANshave been Investigated, less is known about their effects whenpresent as components of mixtures. As the ability to form DNAadducts in vivo Is generally indicative of carcinogenic activityof PAHs, we have compared the DNA binding potencles of dibenzo[a,e]pyrene(DB[a,e]P), dibenzo[a,h]pyrene (DB[a,h]P), dibenzo[a,i]pyrene(DB[a,i]P),dibenzo[a,l]pyrene (DB[a,l]P) and benzo [a]pyrene(B[a]P), when applied topically, either singly or in combination,to the skin of male Parkes mice. DNA isolated from the skinand lungs was analysed by ³²P-postlabelling. The adducts formedby each PAM exhibited markedly different chromato graphic mobifitieson polyethylenelmine-cellulose TLC plates. The relative bindingpotendies of the compounds in both skin and lungs were: DB[a,l]P>B[a]PDB > DB[a,h]P > DB[a,i]P > DB[a,e]P, in good agreementwith their reported carcinogenicitlesin mouse skin. The majorityof adducts were removed from DNA within 21 days of treat ment,but low levels of adducts were found to persist for at least3 months in both tissues. When DB[a,l]P, DB[a,e]P and B[a]Pwere applied together to mouse skin, a total binding 31% lowerthan expected was detected, while with a mixture of DB[a,e]Pand B[a]P the binding to DNA In skin was 65% higher than expectedfrom the binding Levels of the carcino gens when applied singly.Other binary combinations of these three PAils gave adduct levelssimilar to the sum of the binding levels of the individual componentswhen applied singly. The results demonstrate the usefulnessof 32 labelling for the assessment of the DNA binding potenciesof PAHs in mouse tissues, and for the detection of interactionsbetween components of mixtures of carcinogens.     

A quantitative comparison of dibenzo[a,l]pyrene-DNA adduct formation by recombinant human cytochrome P450 microsomes.

Dibenzo[a,l]pyrene (DB[a,l]P), an extremely potent environmental carcinogen, is metabolically activated in mammalian cells and microsomes through the fjord-region dihydrodiol, trans-DB[a,l]P-11, 12-diol, to syn- and anti-DB[a,l]P-11,12-diol-13,14-epoxides (syn- and anti-DB[a,l]PDEs). The role of seven individual recombinant human cytochrome P450s (1A1, 1A2, 1B1, 2B6, 2C9, 2E1, and 3A4) in the metabolic activation of DB[a,l]P and formation of DNA adducts was examined by using (32)P postlabeling, thin-layer chromatography, and high-pressure liquid chromatography. We found that, in the presence of epoxide hydrolase, only P450 1A1 and P450 1B1 catalyzed the formation of DB[a,l]PDE-DNA adducts and several unidentified polar adducts. Human P450 1A1 catalyzed the formation of DB[a, l]PDE-DNA adducts and unidentified polar adducts at rates threefold and 17-fold greater than did human P450 1B1 (256 fmol/h/nmol P450 versus 90 fmol/h/nmol P450 and 132 fmol/h/nmol P450 versus 8 fmol/h/nmol P450, respectively). P450 1A1 DNA adducts were derived from both anti- and syn-DB[a,l]PDE at rates of 73 fmol/h/nmol P450 and 51 fmol/h/nmol P450, respectively. P450 1B1 produced adducts derived from anti-DB[a,l]PDE at a rate of 82 fmol/h/nmol, whereas only a small number of adducts were derived from syn-DB[a,l]PDE (0.4 fmol/h/nmol). These results demonstrated the potential of human P450 1A1 and P450 1B1 to contribute to the metabolic activation and carcinogenicity of DB[a,l]P and provided additional evidence that human P450 1A1 and 1B1 differ in their stereospecific activation of DB[a,l]P. Mol. Carcinog. 26:74-82, 1999. Published 1999 Wiley-Liss, Inc.     

Interspecies differences in the major DNA adducts formed from benzo(a)pyrene but not 7,12-dimethylbenz(a)anthracene in rat and human mammary cell cultures

Mammary epithelial cells from rats and humans show both quantitative and qualitative species- and carcinogen-specific differences in their abilities to activate benzo(a)pyrene (B(a)P) and 7,12-dimethylbenz(a)anthracene (DMBA). Previous studies of the DNA binding of these compounds in mammary epithelial cells demonstrated that rat cells bound relatively more DMBA than B(a)P to DNA under identical treatment conditions, while the opposite pattern was exhibited by human mammary epithelial cells. The specific DNA adducts formed in these cells after 24-h incubations with [3H]DMBA and [3H]B(a)P were analyzed to determine if there were qualitative as well as quantitative differences in the amounts of individual adducts. Similar proportions of specific DMBA-DNA adducts were found in both rat and human cells, although the total amount of adducts formed was significantly higher in the rat cells. In contrast, an essentially qualitative species-specific difference was observed in the major B(a)P-DNA adduct present in the rat and human cells. The major B(a)P adduct formed in the human mammary epithelial cells was identified as the (+)-anti-B(a)P-7,8-dihydrodiol-9, 10-epoxide(BPDE)-deoxyguanosine adduct. However, this adduct was formed at very low levels in the rat mammary epithelial cells. The rat cells contained a large proportion of syn-BPDE adducts, and other unidentified B(a)P-DNA adducts. The high level of the (+)-anti-BPDE-deoxyguanosine adduct in the human but not the rat mammary cells is consistent with the potential role of (+)-anti-BPDE in the high mutagenic activity of B(a)P in the cell-mediated mutagenesis assays using the human mammary cells as activators, and the low mutagenic activity of B(a)P when rat cells were used as activators. The quantitative differences in the activation of DMBA by cells from these two species are also consistent with the cell-mediated mutagenic activities of DMBA using these cells as activators. These results suggest that the higher carcinogenic activity of DMBA compared to B(a)P in the rat mammary gland may not be indicative of the relative carcinogenic potencies of these compounds for human mammary cells.     

Tumor-initiating activity and carcinogenicity of dibenzo[a,l]pyrene versus 7, 12-dimethylbenz[a]anthracene and benzo[a]pyrene at low doses in mouse skin

Dibenzo[a,l]pyrene (DB[a,l]P) is an extremely potent carcinogenthat may be present in environmental samples. Dose-responsestudies were conducted at low doses in mouse skin by initiation-promotionand repeated application to compare its activity to that of7,12-dimethylbenz[a]anthracene (DMBA), benzo[a]pyrene (B[a]P),DB[a,l]P-8,9-dihydrodiol and DB[a,l]P-11,12-dihydrodiol. FemaleSENCAR mice were initiated with 1 or 0.25 nmol of DB[a, l]P,DMBA, B[a]P or DB[a,l]P-11,12-dihydrodiol and promoted withphorbol ester acetate. At 1 nmol, DB[a, l]P induced 2.6 tumors/mouse,whereas DB[a,l]P-11,12-dihydrodiol and DMBA induced 0.17 and0.29 tumors/mouse respectively. At the low dose, DB[a,l]P induced0.79 tumors/mouse, but the other two compounds were virtuallyinactive. B[a]P, tested only at 1 nmol, was inactive. Thesethree compounds, as well as DB[a,l]P-8,9-dihydrodiol, were testedby repeated application twice weekly for 40 weeks at 1 and 4nmol per dose. In addition, DB[a,l]P, DMBA and B[a]P were alsotested at 8 nmol. At 8 and 4 nmol, DB[a,l]P induced malignanttumors in 91 and 70% of mice respectively. At 4 nmol DB[a, l]P-11,12-dihydrodiolelicited only benign tumors in 36% of mice. At 4 nmol DMBA inducedtwo carcinomas in one mouse and at 8 nmol it induced one papillomaand one sebaceous gland adenoma. B[a]P and DB[a,l]P-8,9-dihydrodiolwere inactive at all doses tested. These results demonstratethat DB[a, l]P is a much more potent carcinogen than DMBA, thearomatic hydrocarbon previously considered to be the most potent.Combination of these results with previous comparisons of DB[a,l]P,DB[a,l]P-11,12-dihydrodiol, DMBA and B[a]P at higher doses (E.L.Cavalieri et al. (1991) Carcinogenesis, 12, 1939–1944)shows clearly the interference of toxicity with the tumorigenicityof DB[a,l]P and its 11,12-dihydrodiol.