Comparative in vitro metabolism of benzo[a]pyrene by recombinant zebrafish CYP1A and liver microsomes from beta-naphthoflavone-treated rainbow trout.

The zebrafish (Danio rerio) is a sensitive non-mammalian model used for studying polycyclic aromatic hydrocarbon (PAH)-induced chemical carcinogenesis. The susceptibility of zebrafish to PAH-induced carcinogenesis may be related to the ability of the zebrafish P450s to bioactivate these procarcinogens. As a part of our overall effort to identify the various P450 enzymes that are involved in the activation and detoxification of PAHs in zebrafish, therefore, we have examined the ability of recombinant zebrafish CYP1A (zCYP1A) expressed in yeast to metabolize BaP in vitro. Comparison studies also were conducted with liver microsomes from beta-naphthoflavone (BNF)-treated rainbow trout (Oncorhynchus mykiss). Results demonstrated that the trout liver microsomes were almost twice as active as zCYP1A in oxidizing BaP, with Vmax values of 1.7 and 0.94 nmol/min/nmol P450 for trout and zebrafish preparations, respectively. Like trout CYP1A1, cDNA-expressed zCYP1A was found to oxidize BaP to phenols, quinones and diols (BaP-7,8-diol and BaP-9,10-diol) in the presence of exogenous human microsomal epoxide hydrolase (hEH). BaP-7,8-diol is the precursor of the ultimate carcinogen, BaP-7,8-diol-9,10-epoxide (BaPDE). The ability of zCYP1A to bioactivate BaP was confirmed by the formation of DNA adducts when calf thymus DNA was added to the incubation mixture. BaP-DNA binding was enhanced by the addition of hEH to the incubation mixture. HPLC analysis of the [33P]-postlabeled DNA adducts showed the formation of at least four adducts mediated by both zCYP1A and trout liver microsomes, and one of these adducts co-migrated with BaPDE-dG in HPLC analysis. The addition of hEH to the incubation mixture decreased the formation of BaPDE-dG by zCYP1A and by trout liver microsomes while increasing the formation of an unidentified DNA adduct in the case of zCYP1A. zCYP1A also mediated the binding of BaP to protein, providing further evidence that this enzyme is capable of oxidizing BaP to reactive metabolites that bind to macromolecules. It thus appears that zCYP1A may play an important role in BaP-induced carcinogenesis in the zebrafish model by catalyzing the sequential formation of the ultimate diol epoxide carcinogenic metabolite of BaP.

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.

Effects of the (-)-anti-11R,12S-dihydrodiol 13S,14R-epoxide of dibenzo.

The tumor suppressor protein p53 plays an important role in recognition of DNA damage and induction of subsequent cell cycle arrest. One of its target genes encodes the protein p21(WAF1), which is involved in mediation of growth arrest after DNA damage has occurred. Dibenzo[a,l]pyrene (DB[a,l]P) is a polycyclic aromatic hydrocarbon which is an exceptionally potent carcinogen. A reactive secondary metabolite of DB[a,l]P, the fjord region (-)-anti-11R,12S-dihydrodiol 13R,14S-epoxide [(-)-anti-DB[a,l]PDE] was used to investigate DNA damage via adduct formation and cell cycle arrest in human diploid fibroblast cell cultures (HDF). Synchronous HDF were exposed to increasing concentrations (0.014, 0.028 and 0.07 microM) of (-)-anti-DB[a,l]PDE and at 1, 12, 24 and 42 h after treatment cell pellets were analyzed for DNA adduct formation and cell cycle arrest. Exposure of HDF to 0.07 microM (-)-anti-DB[a,l]PDE caused a total DNA binding level of 113 pmol adducts/mg DNA (42 h after treatment). G(1) arrest was induced by this treatment, with 91% of the cells remaining in G(1) phase compared with the solvent-treated control cultures (50%) as analyzed by propidium iodide staining and flow cytometry. Further investigation of the percentage of cells in S phase by 5-bromo-2'-deoxyuridine incorporation confirmed the G(1) arrest in HDF treated with 0.07 microM (-)-anti-DB[a,l]PDE, with only 1.5% of the cells moving into S phase compared with 39% in the control 42 h after treatment. Induction of p53 and p21(WAF1) was demonstrated by western blot analysis.

Cytochrome P4501B1 mediates induction of bone marrow cytotoxicity and preleukemia cells in mice treated with 7,12-dimethylbenz[a]anthracene.

Humans are exposed to polycyclic aromatic hydrocarbons (PAHs) through many environmental pollutants, especially cigarette smoke. These chemicals cause a variety of tumors and immunotoxic effects, as a consequence of bioactivation by P-450 cytochromes to dihydrodiol epoxides. The recently identified cytochrome P4501B1 (CYP1B1) bioactivates PAHs but is also a physiological regulator, as evidenced by linkage of CYP1B1 deficiency to congenital human glaucoma. This investigation demonstrates that CYP1B1 null mice are almost completely protected from the acute bone marrow cytotoxic and preleukemic effects of the prototypic PAH 7,12-dimethylbenz[a]anthracene (DMBA). CYP1B1 null mice did not produce the appreciable amounts of bone marrow DMBA dihydrodiol epoxide DNA adducts present in wild-type mice, despite comparable hepatic inductions of the prominent PAH-metabolizing P-450 cytochrome, CYP1A1. Wild-type mice constitutively expressed low levels of bone marrow CYP1B1. These findings suggest that CYP1B1 is responsible for the formation of DMBA dihydrodiol epoxides in the bone marrow. Furthermore, this study substantiates the importance of DMBA dihydrodiol epoxide generation at the site of cancer initiation and suggests that tissue-specific constitutive CYP1B1 expression may contribute to cancer susceptibility in the human population.

Formation of stable DNA adducts and apurinic sites upon metabolic activation of bay and fjord region polycyclic aromatic hydrocarbons in human cell cultures.

Carcinogenic polycyclic aromatic hydrocarbons (PAH), such as benzo[a]pyrene (B[a]P), 7,12-dimethylbenz[a]anthracene (DMBA), and dibenzo[a,l]pyrene (DB[a,l]P), are metabolically activated to electrophilically reactive bay or fjord region diol epoxides that bind to the exocyclic amino groups of purine bases in DNA to form stable adducts. In addition, it has been reported that these PAH can be enzymatically oxidized to yield radical cations that form apurinic (AP) sites in DNA via depurinating adducts. The formation of stable adducts and AP sites in DNA of human cells exposed to PAH was examined in cytochrome P450 (P450)-expressing mammary carcinoma MCF-7 cells and in leukemia HL-60 cells, which display a high peroxidase but no P450-mediated activity, after exposure to these PAH. Stable DNA adducts were assessed by (33)P-postlabeling/HPLC analysis, and the induction of AP sites in DNA was analyzed by an aldehyde reactive probe (ARP) and a slot blot method. After exposure for 4 h, the levels of stable DNA adducts were comparable in MCF-7 cells treated with B[a]P and DMBA, but significantly lower than those observed in MCF-7 cells treated with the stronger carcinogen DB[a,l]P. While the levels of stable adducts increased more than 10-fold (B[a]P and DMBA) or 100-fold (DB[a,l]P) after exposure for 24 h, the levels of AP sites remained low after both treatment periods. Thus, the levels of stable adducts were approximately 5-fold higher than the levels of AP sites after treatment with B[a]P or DMBA and more than 100-fold higher in cells exposed to DB[a,l]P for 24 h. None of these carcinogenic PAH formed detectable levels of stable DNA adducts or AP sites in HL-60 cells. The results demonstrate that metabolic activation of B[a]P, DMBA, and DB[a,l]P is catalyzed by P450 enzymes leading to diol epoxides that form predominantly stable DNA adducts but only low levels of AP sites.

Cancer initiation by polycyclic aromatic hydrocarbons results from formation of stable DNA adducts rather than apurinic sites.

Polycyclic aromatic hydrocarbons (PAHs) are ubiquitous environmental pollutants with high carcinogenic potencies that have been linked to the etiology of human cancers through their presence in cigarette smoke and environmental mixtures. They are metabolically activated in cells by cytochrome P450 enzymes and/or peroxidases to reactive intermediates that damage DNA. One pathway of activation forms dihydrodiol epoxides that covalently bind to exocyclic amino groups of purines in DNA to form stable adducts. Another pathway involves formation of radical cations that bind to the N7 or C8 of purines to form unstable adducts that depurinate to leave apurinic (AP) sites in DNA. In the present study the proportions of stable DNA adducts and AP sites formed by the carcinogenic PAHs dibenzo[a,l]-pyrene (DB[a,l]P), 7,12-dimethylbenz[a]anthracene (DMBA), and benzo[a]pyrene (B[a]P) have been investigated in a target tissue for carcinogenesis, mouse epidermis. After topical application of the PAHs on the skin of female SENCAR mice epidermal DNA was isolated and the formation of stable DNA adducts was measured by (33)P-postlabeling and HPLC analysis. AP sites in DNA were measured with an aldehyde reactive probe in a slot-blot assay. At both 4 and 24 h after exposure, DB[a,l]P formed significantly higher amounts of stable DNA adducts than DMBA, and B[a]P exhibited the lowest level of binding. In contrast, the number of AP sites present in mice treated with these PAHs was in the order: DMBA > B[a]P >> DB[a,l]P. The level of AP sites was significantly lower than the level of stable adducts for each PAH. The most potent carcinogen, DB[a,l]P, induced the highest level of stable adducts and the lowest level of AP sites in epidermal DNA. These results indicate that stable DNA adducts rather than AP sites are responsible for tumor initiation by carcinogenic PAHs.

Inhibition of dibenzo[a,l]pyrene-induced multi-organ carcinogenesis by dietary chlorophyllin in rainbow trout.

Cancer chemoprevention by dietary chlorophyllin (CHL) was investigated in a rainbow trout multi-organ tumor model. In study 1, duplicate groups of 130 juvenile trout were treated for 2 weeks with control diet, 500 p.p.m. dibenzo[a,l]pyrene (DB[a,l]P) or 500 p.p.m. DB[a,l]P + 2052 p.p.m. CHL, then returned to control diet. DB[a,l]P alone proved somewhat toxic but induced high tumor incidences in liver (61%), stomach (91%) and swimbladder (53%) 11 months after initiation. CHL co-feeding abrogated DB[a,l]P acute toxicity and reduced tumor incidences to 18% in liver, 34% in stomach and 3% in swimbladder (P

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.

The level of DNA modification by (+)-syn-(11S,12R,13S,14R)- and (-)-anti-(11R,12S,13S,14R)-dihydrodiol epoxides of dibenzo[a,l]pyrene determined the effect on the proteins p53 and p21WAF1 in the human mammary carcinoma cell line MCF-7.

The polycyclic aromatic hydrocarbon (PAH) dibenzo[a,l]pyrene (DB[a,l]P), the most carcinogenic PAH tested in rodent bioassays, exerts its pathobiological activity via metabolic formation of electrophilically reactive DNA-binding fjord region (+)-syn-(11S,12R,13S,14R)- or (-)-anti-(11R,12S,13S,14R)-DB[a,l]P-dihydrodiol epoxides (DB[a,l]-PDEs). DB[a,l]P is metabolized to these DB[a,l]PDEs which bind to DNA in human mammary carcinoma MCF-7 cells. The molecular response of MCF-7 cells to DNA damage caused by DB[a,l]PDEs was investigated by analyzing effects on the expression of the tumor suppressor protein p53 and one of its target gene products, the cyclin-dependent kinase inhibitor p21WAF1. Treatment of MCF-7 cells with (+)-syn- and (-)-anti-DB[a,l]PDE at a concentration range of 0.001-0.1 microM resulted in DB[a,l]PDE-DNA adduct levels between 2 and 30, and 3 and 80 pmol/mg DNA, respectively, 8 h after exposure. (-)-anti-DB[a,l]PDE exhibited a higher binding efficiency that correlated with a significantly stronger p53 response at low concentrations of the dihydrodiol epoxides. The level of p53 increased by 6-8 h after treatment. The p21WAF1 protein amount exceeded control levels by 12 h and remained elevated for 96 h. At a dose of 0.01 microM (+)-syn-DB[a,l]PDE, an increase in p21WAF1 was observed in the absence of a detectable change in p53 levels. The results indicate that the increase in p53 induced by DB[a,l]PDEs in MCF-7 cells requires an adduct level of approximately 15 pmot/mg DNA and suggest that the level of adducts rather than the specific structure of the DB[a,l]PDE-DNA adduct formed triggers the p53 response. The PAH-DNA adduct level formed may determine whether p53 and p21VAF1 pathways respond, resulting in cell-cycle arrest, or fail to respond and increase the risk of mutation induction by these DNA lesions.

Metabolic activation of 4H-cyclopenta[def]chrysene in human mammary carcinoma MCF-7 cell cultures.

The tumor initiating activities of 4H-cyclopenta[def]chrysene (C[def]C) and its two putative reactive metabolites, trans-1, 2-dihydroxy-anti-3,3a-epoxy-1,2,3, 3a-tetrahydro-4H-cyclopenta[def]chrysene (C[def]C-3,3a-DE) and trans-6,7-dihydroxy-anti-8,9-epoxy-6,7,8, 9-tetrahydro-4H-cyclopenta[def]chrysene (C[def]C-8,9-DE), were evaluated previously in mice [Amin, S., et al. (1995) Carcinogenesis 16, 2813-2817]. C[def]C-3,3a-DE was the more active inducer of lung tumors and elicited twice as many tumors as C[def]C-8,9-DE. In this study, the route of metabolism of C[def]C to DNA-reactive metabolites in the human mammary carcinoma cell line (MCF-7) was investigated using the 32P-postlabeling assay. The results show that metabolic activation to DNA-binding species proceeds through the formation of both trans-1,2-dihydrodiol and trans-6,7-dihydrodiol metabolites of C[def]C. At a 1 microM dose, adducts from the methylene-bridged (C[def]C-3,3a-DE) and bay region (C[def]C-8,9-DE) dihydrodiol epoxides were detected in comparable amounts. In contrast, the majority of the postlabeled adducts recovered from cells exposed to a 10 microM dose were derived from the bay region dihydrodiol epoxide, C[def]C-8,9-DE. Using markers from reactions of the dihydrodiol epoxides with deoxyguanosine 3'-phosphate and deoxyadenosine 3'-phosphate, it was shown that the major radioactive spots formed with both anti-C[def]C-3,3a-DE and anti-C[def]C-8,9-DE chromatographed with deoxyguanosine adduct markers. Thus, the human cells used in these studies can activate C[def]C to carcinogenic metabolites.

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.

Stable expression of human cytochrome P450 1B1 in V79 Chinese hamster cells and metabolically catalyzed DNA adduct formation of dibenzo[a,l]pyrene.

Chinese hamster V79 cell lines were constructed for stable expression of human cytochrome P450 1B1 (P450 1B1) in order to study its role in the metabolic activation of chemicals and toxicological consequences. The new V79 cell lines were applied to studies on DNA adduct formation of the polycyclic aromatic hydrocarbon (PAH) dibenzo[a,l]pyrene (DB[a,l]P). This compound has been found to be an environmental pollutant, and in rodent bioassays it is the most carcinogenic PAH yet discovered. Activation of DB[a,l]P in various metabolizing systems occurs via fjord region DB[a,l]P-11, 12-dihydrodiol 13,14-epoxides (DB[a,l]PDE): we found that DB[a,l]P is stereoselectively metabolized in human mammary carcinoma MCF-7 cells to the (-)-anti- and (+)-syn-DB[a,l]PDE which both bind extensively to cellular DNA. To follow up this study and to relate specific DNA adducts to activation by individual P450 isoforms, the newly established V79 cells stably expressing human P450 1B1 were compared with those expressing human P450 1A1. DNA adduct formation in both V79 cell lines differed distinctively after incubation with DB[a,l]P or its enantiomeric 11,12-dihydrodiols. Human P450 1A1 catalyzed the formation of DB[a,l]PDE-DNA adducts as well as several highly polar DNA adducts as yet unidentified. The proportion of these highly polar adducts to DB[a,l]PDE adducts was dependent upon both the concentration of DB[a,l]P and the time of exposure. In contrast, V79 cells stably expressing human P450 1B1 generated exclusively DB[a,l]PDE-DNA adducts. Differences in the total level of DNA binding were also observed. Exposure to 0.1 microM DB[a,l]P for 6 h caused a significantly higher level of DNA adducts in V79 cells stably expressing human P450 1B1 (370 pmol/mg of DNA) compared to those with human P450 1A1 (35 pmol/mg of DNA). A 4-fold higher extent of DNA binding was catalyzed by human P450 1B1 (506 pmol/mg of DNA) compared to human P450 1A1 (130 pmol/mg of DNA) 6 h after treatment with 0.05 microM (-)-(11R,12R)-dihydrodiol. In cells stably expressing human P450 1B1 the DNA adducts were derived exclusively from the (-)-anti-DB[a,l]PDE. These results indicate that human P450 1B1 and P450 1A1 differ in their regio- and stereochemical selectivity of activation of DB[a,l]P with P450 1B1 forming a higher proportion of the highly carcinogenic (-)-anti-(11R, 12S,13S,14R)-DB[a,l]PDE metabolite.

Stereoselective activation of dibenzo[a,l]pyrene and its trans-11,12-dihydrodiol to fjord region 11,12-diol 13,14-epoxides in a human mammary carcinoma MCF-7 cell-mediated V79 cell mutation assay.

Dibenzo[a,l]pyrene (DB[a,l]P) represents the most potent carcinogenic polycyclic aromatic hydrocarbon (PAH) yet discovered. Like other PAHs, DB[a,l]P requires metabolic activation to exert its mutagenic and/or carcinogenic activity. In the human mammary carcinoma cell line MCF-7, DB[a,l]P is stereoselectively metabolized to the (-)-anti- and (+)-syn-DB[a,l]P-11,12-diol 13,14-epoxides (DB[a,l]PDE) which both bind extensively to deoxyadenosine residues in DNA. To further characterize the underlying mechanism of its strong carcinogenicity, the relationship between DNA binding and mutagenicity of DB[a,l]P was determined. Racemic DB[a,l]P-11,12-dihydrodiol and the two individual (+)- and (-)-enantiomers, the metabolic precursors of the stereoisomeric fjord region dihydrodiol epoxides, were also investigated. Induction of mutations at the HPRT locus was measured in a MCF-7 cell-mediated Chinese hamster V79 cell mutation assay. The parent hydrocarbon, (+/-)-DB[a,l]P-11,12-dihydrodiol, and (-)-DB[a,l]P-11,12-dihydrodiol were highly mutagenic under the assay conditions. In contrast, (+)-DB[a,l]P-(11S,12S)-dihydrodiol was not mutagenic using MCF-7 cells as the metabolic activating system. Analysis of DNA adducts in the same experiments revealed that MCF-7 cells treated with (-)-DB[a,l]P-11,12-dihydrodiol formed exclusively (-)-anti-DB[a,l]-PDE adducts whereas cells treated with (+)-DB[a,l]P-11,12-dihydrodiol did not contain detectable levels of DNA adducts. These results suggest that specific cytochrome P450 enzymes may have high stereoselectivity for activation of the two DB[a,l]P-11,12-dihydrodiol enantiomers, and this may play an important role in the metabolic activation of the strong carcinogen DB[a,l]P in human cells.

Role of cytochrome P450 enzyme induction in the metabolic activation of benzo[c]phenanthrene in human cell lines and mouse epidermis.

The environmental contaminant benzo[c]phenanthrene (B[c]Ph) has weak carcinogenic activity in rodent bioassays; however, the fjord region diol epoxides of B[c]Ph, B[c]Ph-3,4-diol 1,2-epoxides (B[c]PhDE), are potent carcinogens. To determine the role of cytochrome P450 isozymes in the activation of B[c]Ph in MCF-7 cells and the low activation of B[c]Ph in mouse skin, cells of the MCF-7 and the human hepatoma HepG2 cell lines were treated with the potent Ah receptor agonist 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) prior to exposure to B[c]Ph for 24 h. Mice were treated topically with 1 microg of TCDD or vehicle (control) for 73 h and then with 2 micromol of B[c]Ph for 24 h. In MCF-7 cells, TCDD exposure increased B[c]PhDE-DNA adduct levels more than 3-fold with a 10-fold increase in the (-)-B[c]PhDE-2-dA(t) adduct. Treatment of HepG2 cells with TCDD prior to B[c]Ph application did not increase B[c]PhDE-DNA binding. Total B[c]PhDE-DNA adducts increased 3-fold in TCDD-treated mouse epidermis: the majority of the increase resulted from (+)-B[c]PhDE-1-dA adducts. Analysis of P450 enzymes by Western blotting detected a large increase of P4501B1 but almost no increase in P4501A1 in MCF-7 cells exposed to 10 microM B[c]Ph for 24 or 48 h. In HepG2 cells, there were no detectable levels of P4501A1 or P4501B1 after treatment with 10 microM B[c]Ph for 24 h. In contrast, topical application of 2 micromol of B[c]Ph to mouse skin for 48 or 72 h increased P4501A1, but no P4501B1 was detected. As a measure of P450 activity, the metabolism of 7,12-dimethylbenz[a]anthracene (DMBA) was analyzed in microsomes prepared from MCF-7 and HepG2 cells exposed to 0.1% DMSO, 10 microM B[c]Ph, or 10 nM TCDD for 24 or 48 h and from mouse epidermis treated with 1 microg of TCDD, or vehicle control for 72 h, or 2 micromol of B[c]Ph for 48 h. The levels of DMBA metabolites were low or undetectable in microsomes from B[c]Ph-treated MCF-7 and HepG2 cells, but a metabolite pattern consistent with P4501A1 metabolism of DMBA was present in B[c]Ph-exposed mouse epidermal microsomes. TCDD-treated MCF-7 cells, HepG2 cells, and mouse epidermis had DMBA metabolism patterns characteristic of P4501A1 activity. Microsomes from TCDD-treated human cells formed a higher proportion of the proximate carcinogenic metabolite DMBA-3,4-dihydrodiol (16% of total identified metabolites) than TCDD-treated mouse epidermis (2%). In mouse epidermis, the weak ability of B[c]Ph to increase hydrocarbon-metabolizing activity and the increase in mainly P4501A1, leading to formation of the less carcinogenic stereoisomer B[c]PhDE-1, may explain the low carcinogenic activity of B[c]Ph. In a human mammary carcinoma cell line, treatment with B[c]Ph increases mainly P4501B1 and results in formation of a higher proportion of the more carcinogenic B[c]PhDE-2. This indicates that cells in which B[c]Ph treatment increases P4501B1 levels effectively activate B[c]Ph to potent carcinogenic metabolites.

Metabolic activation of benzo[g]chrysene in the human mammary carcinoma cell line MCF-7.

Benzo[g]chrysene (BgC) is an environmental pollutant, and recent studies have demonstrated that anti- BgC-11,12-dihydrodiol 13,14-epoxide (anti-BgCDE) is a potent mammary carcinogen in rats. To determine whether BgC can be metabolically activated to anti-BgCDE in human cells, the human mammary carcinoma cell line MCF-7 was treated with BgC and with the racemic trans-3,4- and 11,12-dihydrodiols. The DNA adducts formed in these experiments were examined using 32P-postlabeling, and specific adducts were identified through comparisons with adducts obtained by the reaction of the racemic syn- and anti-BgCDEs with calf thymus DNA and with purine deoxyribonucleoside-3'-phosphates in vitro. It was found that BgC is metabolically activated in MCF-7 cells to form major DNA adducts through both the syn- and anti-11,12-dihydrodiol 13,14-epoxide metabolites. BgC is therefore a potential environmental risk to humans. The major BgC-DNA adducts formed from both the dihydrodiol-epoxide diastereomers were deoxyadenosine adducts. Thus, BgC has DNA-binding properties that are very similar to those of the potent mammary carcinogens 7,12-dimethylbenz[a]anthracene and dibenzo[a,l]pyrene.

Formation of stable adducts and absence of depurinating DNA adducts in cells and DNA treated with the potent carcinogen dibenzo[a,l]pyrene or its diol epoxides.

Polycyclic aromatic hydrocarbons (PAH) are widespread environmental contaminants, and some are potent carcinogens in rodents. Carcinogenic PAH are activated in cells to metabolites that react with DNA to form stable covalent DNA adducts. It has been proposed [Cavalieri, E. L. & Roger, E. G. (1995) Xenobiotica 25, 677-688] that unstable DNA adducts are also formed and that apurinic sites in the DNA resulting from unstable PAH adducts play a key role in the initiation of cancer. The potent carcinogen dibenzo[a,l]pyrene (DB[a, l]P) is activated in cells to (+)-syn- and (-)-anti-DB[a,l]P-11, 12-diol-13,14-epoxide (DB[a,l]PDE), which have been shown to form stable adducts with DNA. To evaluate the importance of unstable PAH adducts, we compared stable adduct formation to apurinic site formation. Stable DB[a,l]PDE adducts were determined by 33P-postlabeling and HPLC. To measure apurinic sites they were converted to strand breaks, and these were monitored by examining the integrity of a particular restriction fragment of the dihydrofolate reductase gene. The method easily detected apurinic sites resulting from methylation by treatment of cells or DNA with dimethyl sulfate or from reaction of DNA with DB[a,l]P in the presence of horseradish peroxidase. We estimate the method could detect 0.1 apurinic site in the 14-kb fragment examined. However, apurinic sites were below our limit of detection in DNA treated directly with (+)-syn- or (-)-anti-DB[a,l]PDE or in DNA from Chinese hamster ovary B11 cells so treated, although in these samples the frequency of stable adducts ranged from 3 to 10 per 14 kb. We also treated the human mammary carcinoma cell line MCF-7 with DB[a,l]P and again could not detect significant amounts of unstable adducts. These results indicate that the proportion of stable adducts formed by DB[a,l]P activated in cells and its diol epoxides is greater than 99% and suggest a predominant role for stable DNA adducts in the carcinogenic activity of DB[a,l]P.

Benzo[c]phenanthrene is activated to DNA-binding diol epoxides in the human mammary carcinoma cell line MCF-7 but only limited activation occurs in mouse skin.

Benzo[c]phenanthrene (B[c]Ph) is an environmental contaminant with low carcinogenic activity in rodent bioassays. B[c]Ph-3,4-diol-1,2-epoxides (B[c]PhDE), however, are among the most tumorigenic diol epoxides known. To determine whether human cells are capable of activating B[c]Ph to DNA-binding metabolites, cultures of the human mammary cell line, MCF-7, were exposed to 10 microM B[c]Ph for 48, 72 and 96 h or to 1 microM (+/-)-B[c]Ph-3,4-dihydrodiol for 48 h. The B[c]Ph-DNA adducts were analyzed by 33P-postlabeling and reverse-phase HPLC. The major B[c]Ph-DNA adducts were formed by the trans-addition of (4R,3S)-dihydroxy-(2S,1R)-epoxy-1,2,3,4-tetrahydro-B[c]Ph to deoxyadenosine [(-)-B[c]PhDE-2dAt] and by the cis- and trans-addition of (4S,3R)-dihydroxy-(2S,1R)-epoxy-1,2,3,4-tetrahydro-B[c]Ph to deoxyadenosine [(+)-B[c]PhDE-1dAc and (+)-B[c]PhDE-1dAt]. Smaller amounts of the trans-addition of (-)-B[c]PhDE-2 were bound to deoxyguanosine. To determine whether B[c]Ph can be metabolically activated to diol epoxides in mouse epidermis, female SENCAR mice were treated topically with 2 micromol B[c]Ph for 24, 48 or 72 h or with 0.4 micromol (+/-)-B[c]Ph-3,4-dihydrodiol for 24 or 48 h. In B[c]Ph-treated mice, only small amounts of three B[c]PhDE-DNA adducts were detected [(-)-B[c]PhDE-2dAt, (+)-B[c]PhDE-1dAt and (+)-B[c]PhDE-1dAc] at 24, 48 and 72 h. In contrast, mice treated topically with 0.4 micromol (+/-)-B[c]Ph-3,4-dihydrodiol formed B[c]PhDE-DNA adducts at levels 6-fold greater than those observed with B[c]Ph at 48 h. The higher formation of B[c]PhDE-DNA adducts by (+/-)-B[c]Ph-3,4-dihydrodiol correlates with the greater potency of (+/-)-B[c]Ph-3,4-dihydrodiol than of B[c]Ph as a tumor initiator in mouse skin. The low extent of formation of B[c]PhDE from B[c]Ph in mouse epidermis may explain the low tumorigenicity of B[c]Ph in this tissue. These results indicate activation of B[c]Ph in mouse skin and tumorigenesis results in that tissue may not adequately assess the potential capability of cells from humans to activate B[c]Ph to ultimate carcinogenic metabolites.

Stereoselective activation of dibenzo[a,l]pyrene to (-)-anti (11R,12S,13S,14R)- and (+)-syn(11S,12R,13S,14R)-11,12-diol-13,14-epoxides which bind extensively to deoxyadenosine residues of DNA in the human mammary carcinoma cell line MCF-7.

Dibenzo[a,l]pyrene (DB[a,l]P) is an environmental contaminant and a very potent carcinogen. DB[a,l]P exceeds the carcinogenic potency of both benzo[a]pyrene and 7,12-dimethylbenz[a]anthracene in rodent bioassays. Previous studies demonstrated that DB[a,l]P is metabolized to DB[a,l]P-11,12-diol-13,14-epoxide (DB[a,l]PDE) in the human mammary carcinoma cell line MCF-7. In the present study the major DNA adducts formed in DB[a,l]P-treated MCF-7 cells have been identified through the use of 33P-postlabeling. TLC and HPLC. DB[a,l]P is metabolically activated in MCF-7 cells to form large amounts of three major DNA adducts and smaller amounts of three other adducts. The three major DNA adducts are with deoxyadenosine: two are formed by reaction of (+)-syn-DB[a,l]PDE (11S,12R,13S,14R), the third by reaction of (-)-anti-DB[a,l]PDE (11R,12S,13S,14R). The results demonstrate that DB[a,l] is stereoselectively metabolized in MCF-7 cells to form one enantiomer of each diol epoxide diastereomer; (+)-syn-DB[a,l]PDE and (-)-anti-DB[a,l]PDE. The high extent of binding of these diol epoxides to deoxyadenosine in DNA of MCF-7 cells may help to explain the very high carcinogenic potency of DB[a,l]P and suggests that DB[a,l]P could also pose a carcinogenic threat to humans.

Stereoselectivity of activation of 7,12-dimethylbenz[a]anthracene- 3,4-dihydrodiol to the anti-diol epoxide metabolite in a human mammary carcinoma MCF-7 cell-mediated V79 cell mutation assay.

7,12-Dimethylbenz[a]anthracene (DMBA), one of the most carcinogenic polycyclic aromatic hydrocarbons in rodent bioassays, is metabolically activated in many tissues to "bay-region" DMBA-3,4-diol-1,2-epoxides (DMBADE). Unlike benzo[a]pyrene, for which the high biological activity of the (7R,8S)-diol-(9S,10R)-epoxide has been established, the low chemical stability of anti-DMBADE has made it impossible to evaluate the role of specific stereoisomers in the biological activity of DMBA. In order to characterize the role of formation of DMBADE diastereomers in the induction of mutations, postlabeling assays using [35S]phosphorothioate with adduct separation by HPLC and immobilized boronate chromatography analyses were developed to allow separation and quantitation of DNA adducts formed from each stereoisomer of DMBADE. In DMBA-treated hamster embryo cell cultures, large quantities of three major adducts (anti-DMBADE-deoxyguanosine, anti-DMBADE-deoxyadenosine, and syn-DMBADE-deoxyadenosine) along with five minor adducts were completely resolved and quantitated. The DNA isolated from a human mammary carcinoma MCF-7 cell-mediated V79 cell mutation assay treated with increasing doses of racemic DMBA-3,4-dihydrodiol contained large amounts of two anti-DMBADE-DNA adducts. The anti-DMBADE adducts accounted for more than 90% of the total adducts at all doses. The number of 6-thioguanine-resistant mutants was proportional to the amount of anti-DMBADE-DNA adducts.(ABSTRACT TRUNCATED AT 250 WORDS)