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.     

Synthesis and tumor-initiating activity in mouse skin of dibenzo[a,l]pyrene syn- and anti-fjord-region diolepoxides

Dibenzo[a,l]pyrene (DB[a,l]P) is the most potent carcinogenamong polycycic aromatic hydrocarbons. Because the fjord-regiondiolepoxide (DE) pathway is one of the mech anisms of activation,(±)-trans-DB [a,l]P-11,12-dihydrodiol,(±)-anti-DB[a,l]PDEand (±)-syn-DB[a,l]PDE were synthesized. The key intermediatefor these syntheses, 12-methoxy-DB[a,l]P, was successfully obtainedby cyclizatlon of 6-(3-methoxybenzyl)benzanthrone with methanesulfonicacid, which in turn was prepared by 1,4 conjugate addition of3-methoxybenzyl magnesium bromide to benzanthrone. The presenceof the DB[a,l]P nucleus in the dihydrodiol epoxides and diolepoxideswas proven by conversion of 12-methoxyDB[a,l]P into the parentcompound in several steps. The tumor-initiating activity ofthe two diolepoxides in mouse skin was compared to that of DB[a,l]P-11,12-dihydrodioland the parent DB[a,l]P Groups of 24 8 week old female SENCARmice were topically initiated with 12, 4 or 133 nmol of compoundIn 100 µl of acetone. Starting 1 week later, promotionwith 12-O-tetradecanoylphorbol-13-acetate (1.62 nmol In 100µl acetone) was begun and continued twice weekly for 30weeks. At the 12, 4 and 1.33 nmol doses, anti-DB[a,l]PDE induced2.0, 0.7 and 0.7 tumors per mouse (t/m) respectively, whereassyn DB[a,l]PDE induced 1.8, 1.5 and 1.8 t/m. At the same threedoses, DB[a,l]P-11,12-dihydrodiol induced 4.6, 4.3 and 2.8 t/m,and DB[a,l]P resulted in 9.3,7.1 and 5.2 t/m. These resultsconfirm that DB[a,l]P is more potent than its 11,12-dihydrodloland show that the two diolepoxides are less tumorlgenic thantheir precursors. At the medium and low doses, syn-DB[a,l]PDEis more tumorigenic than its congener anti-DB[a,l]PDE.     

Comparison of the tumor-initiating activity of 7,12-dimethylbenz[a]anthracene and benzo[a]pyrene in female SENCAR and CD-1 mice

The derivation of mice resistant and susceptible to skin tumorigenesisusing the initiation-promotion regimen is described. Dose-responserelationships for tumor-initiating activities of 7,12-dimethylbenz[a]-anthracene(DMBA) and benzo[a]pyrene (BP) in the susceptible line (SENCAR)are presented. A single topical dose of either 0.1, 1.0, 10or 100 nmol DMBA, followed one week later by twice weekly applicationsof 8.5 nmol 12–0-tetradecanoylphorbol-13-acetate (TPA)for 19 weeks, produced 0, 3.3, 4.9 and 23.1 papillomas per mouse,respectively. Single topical initiating doses of either 50,100 or 200 nmol BP produced 1.7, 3.8 or 7.8 papillomas per mouse,respectively, after 28 weeks of promotion with 8.5 nmol TPA.SENCAR mice were compared with CD-1 mice for the initiatingactivity of DMBA and BP. Initiating doses of 0.1, 1.0, 10 and100 nmol DMBA produced 0.6, 3.8, 7.0 and 24 papillomas per mouse,respectively, in SENCAR mice and in CD-1 mice produced 0, 0.2,3.0 and 5.6 papillomas per mouse, respectively, after 25 weeksof promotion with TPA. With BP as the initiator, 10, 50, 100and 200 nmol doses produced 0.9, 1.6, 3.8 and 8.3 papillomasper mouse, respectively, in SENCAR mice and in CD-1 mice produced0.1, 0.7,1.8 and 3.8 papillomas per mouse, respectively, after25 weeks of promotion with TPA. SENCAR mice were compared with CD-1 mice for possible differencesin the oxidative metabolism of DMBA using epidermal homogenatesas the enzyme source. Basal levels of monooxygenase activitytoward DMBA were similar in both mouse stocks. Epidermal monooxygenaseactivities following pre-treatment with inducers including DMBA,3-methylcholanthrene, dibenz[a,c]anthracene, Aroclor 1254 and2,3,7,8-tetrachlorodibenzo-p-dioxin, also were quite similarin both mouse stocks. High-pressure liquid chromatographic profilesof ethyl acetate/ acetone (2:1) extractable metabolites revealeda close similarity in the patterns as well as the rates of formationof specific metabolites. Metabolites of DMBA tentatively identifiedbased on cochromato-graphy with purified reference standardsincluded phenols, 12-hydroxymethyl-7-methylbenz[a]anthracene,7-hydroxymethyl-12-methylbenz[a]antnracene, 7,12-dihydroxymethylbenz[a]anthracene,(±)-trans-8,9-dihydro-8,9-dihydroxy-7,12-dimethylbenz[a]anthraceneand (±)-trans-5,6-dihydro-5,6-dihydroxy-7,12-dimethylbenz[a]anthracene.The results suggested that differences in oxidative metabolismof DMBA were not responsible for the differences in sensitivityto tumor-initiation between SENCAR and CD-1 mice.     

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.      

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.     

Mutagenesis and carcinogenesis induced by dibenzo[a,l]pyrene in the mouse oral cavity: a potential new model for oral cancer

Cancer of the oral cavity is a serious disease, affecting about 30,000 individuals in US annually. There are several animal models of oral cancer, but each has certain disadvantages. As a new model, we investigated whether topical application of the tobacco smoke carcinogen, dibenzo[a,l]pyrene (DB[a,l]P) is mutagenic and carcinogenic in the oral cavity of the B6C3F1 lacI and B6C3F1 mouse, respectively. B6C3F1 lacI mice received DB[a,l]P (0, 3, 6, 12 nmol) 3× per week. B6C3F1 mice received the same doses and also 24 nmol. At 38 weeks mutagenesis was measured in oral tissues in lacI mice. For the high dose group, the mutant fraction (MF) in upper mucosa and tongue increased about twofold relative to that in vehicle-alone. The increases were statistically significant. The mutational profile in the DB[a,l]P-induced mutants was compared with that induced by benzo[a]pyrene (BaP) in oral tissue. BaP is mutagenic in many tissues when administered by gavage. The mutational profile for DB[a,l]P was more similar to that reported for p53 mutations in head and neck cancers than was that of BaP. At 47 weeks, oral squamous cell carcinomas (OSCC) were found in 31% of the high-dose B6C3F1 group. Elevations of p53 and COX-2 protein were observed in tumor and dysplastic tissue. As DB[a,l]P induces mutations and tumors in the oral cavity, and has a mutational profile in oral tissue similar to that found in p53 in human OSCC, the treatment protocol described here may represent a new and relevant model for cancer of the oral cavity.     

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.     

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.     

Metabolic activation of the potent carcinogen dibenzo[a, l]pyrene by human recombinant cytochromes P450, lung and liver microsomes

The metabolic activation of dibenzo[a, l]pyrene (DB[a, l]P),recently considered the most potent carcinogen among all polycyclicaromatic hydrocarbons, to the 11, 12-dihydrodiol, a precursorof the ultimate carcinogens, the 11, 12-diol-13, 14-epoxides,was investigated using eleven human recombinant cytochrome P450s,as well as human lung and liver microsomes. Of all human P450s,1A1 was the most active in the metabolism of DB[a, l]P (310pmol/min, nmol P450) and had 5–23-fold higher catalyticactivity than other P450s examined. The order of activity inthe formation of the 11, 12-dihydrodiol was as follows: 1A1(116 pmol/min, nmol P450) > 2C9 (29) > 1A2 (22) > 2B6(18) > 3A4 (16) > others (     

Potent inhibitory effects of suicide inhibitors of P450 isozymes on 7,12-dimethylbenz[a]anthracene and benzo[a]pyrene initiated skin tumors

A single dose of 1-ethynylpyrene (EP), 1-vinylpyrene (VP) or 2-ethynylnaphthalene (EN) was applied to the skin of SENCAR mice 5 min before an initiating dose of 7,12-dimethylbenz[a]anthracene (DMBA) or benzo[a]pyrene (B[a]P) and the development of skin tumors then promoted with biweekly topical applications of 12-O-tetradecanoylphorbol-13-acetate (TPA). The application of EP strongly inhibited the formation of skin tumors initiated by either DMBA or B[a]P in a dose-dependent manner. Application of 44 pmol of EP inhibited tumor initiation by 10 nmol of DMBA approximately 25%; application of 440 nmol of EP inhibited tumor initiation by 200 nmol of B[a]P approximately 51%. A high single dose of EP (4.4-44 mumol) nearly eliminated skin tumor initiation by either 10 nmol of DMBA or 200 nmol of B[a]P. Application of VP also inhibited the formation of skin tumors initiated by either DMBA or B[a]P in a dose-dependent manner, but higher doses of VP than of EP were required to produce comparable inhibitions. Application of 44 nmol of VP inhibited tumor initiation by 10 nmol of DMBA approximately 30%; application of 4.4 mumol of VP inhibited tumor initiation by 200 nmol of B[a]P approximately 56%. Application of EN yielded contrasting results. EN inhibited the formation of skin tumors initiated by 10 nmol of DMBA, but the observed dose-dependence was minimal; tumors were decreased about 40% by 3.3 mumol of EN and only about 65% by 132 mumol of EN. A high single dose of EN (132 mumol) increased both the mean number of tumors per mouse and the percentage of mice that developed tumors after initiation by 200 nmol of B[a]P. Topical application of 4.4 mumol of EP, 22 mumol of VP or 33 mumol of EN to the skin of SENCAR mice 5 min before a single initiation dose of 2.5 mumol of N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) had a minimal inhibitory effect (14-28%) on the development of skin tumors produced by subsequent biweekly promotion with TPA. A single dose of 44 mumol of EP or 132 mumol of EN followed by biweekly applications of TPA did not produce skin tumors; however, a dose of 44 mumol of VP followed by promotion with TPA produced a low but significant number of skin tumors.(ABSTRACT TRUNCATED AT 400 WORDS)     

Tumor-initiating activity of the (+)-(S,S)- and (-)-(R,R)- enantiomers of trans-11,12-dihydroxy-11,12-dihydrodibenzo[a,l]pyrene in mouse skin.

A single administration of enantiomerically pure 11,12-dihydrodiols of dibenzo[a,l]pyrene (DB[a,l]P) on the back of NMRI mice and subsequent chronic treatment with 12-O-tetradecanoylphorbol 13-acetate (TPA) (initiation/promotion assay) revealed strikingly different carcinogenic activities of both enantiomers. Tumor-initiating activity of (-)-(11R,12R)-DB[a,l]P-dihydrodiol, which is the metabolic precursor of the (-)-anti-(11R,12S)-dihydrodiol (13S,14R)-epoxide, was exceptionally higher than the corresponding effect of (+)-(11S,12S)-DB[a,l]P-dihydrodiol, the metabolic precursor of (+)-syn-(11S,12R)-dihydrodiol (13S,14R)-epoxide. After topical application of 10 nmol (-)-11,12-dihydrodiol and promotion with TPA twice weekly for a further 18 weeks 93% of treated animals exhibited four to five tumors. In contrast, no neoplasms were observed after treatment with 10 nmol (+)-11,12-dihydrodiol, whereas in the group exposed to 20 nmol of this enantiomer only 13% of mice developed neoplasms (0.1 tumors/survivor). For DB[a,l]P, considered as the most potent carcinogenic polycyclic aromatic hydrocarbon to date, stereoselective formation of (+)-syn- and (-)-anti-11,12-dihydrodiol 13,14-epoxides via the corresponding enantiomeric 11,12-dihydrodiols has been found to be the principal metabolic activation pathway leading to DNA adducts and mutagenicity. Our study demonstrates that the striking difference in carcinogenic activity in mouse skin of (+)-(11S,12S)- and (-)-(11R,12R)-DB[a,l]P-dihydrodiol convincingly reflects the different genotoxicity, i.e. DNA binding and mutagenicity, of both enantiomers observed earlier.     

Synthesis and mutagenicity of the diastereomeric fjord-region 11,12-dihydrodiol 13,14-epoxides of dibenzo [a,l]pyrene

Extensive tumongenicity studies in rodents revealed that dibenzo[a,l]pyrene(DB[a,l]P) is the most potent carcinogen among all polycyclicaromatic hydrocarbons (PAHs) tested so far. The structure ofthe genotoxic metabolite(s) responsible for this exceptionalcarcinogenicity is. unknown. The fjord-region syn- and anti-DB[a,l]P-11,12-dihydrodiol 13,14-epoxides (syn- and anti-DB[a,l]PDE)were synthesized to clarify their role as possible ultimatemutagenic and carcinogenic metabolites of DB [a,l]P.9-Formyl-11,12-dime-thoxybenzo [g] chrysense was prepared from 9-phenanthryl- aceticacid by a photochemical route. After reaction of the aldehydewith trimethylsulfonium iodide to generate an oxiranyl side-chain,treatment with boron trifluoride produced the key intermediate11,12-dimethoxy.DB[a,l]P in 14% overall yield. From 11,12-dimethoxy-DB[a,l]Pthe syn- and anti-DB[a,l]PDE were stereoselectively preparedvia the trans-11,12-dihydrodiol. The mutagenicity of the synand anti-DB[a,l]PDE was examined in four his^– strainsof Salmonella typhimurium and in Chinese hamster V79 cells.In all five test systems, the new dihydrodiolepoxides were morepotent than any of the previously investigated dihydrodlolepoxides.The specific mutagemcity observed with anti-DB[a,l]PDE in strainTA104 exhibited the highest value ever found with any compoundin any his strains of S.typhimurium. The same appears to hetrue for the activity observed with this compound in V79 cells.In all five systems, syn-DB[a,l]PDE was only moderately lessactive than its anti-dlastereomer (     

Effect of ellagic acid and hydroxylated flavonoids on the tumorigenicity of benzo[a]pyrene and ({+/-})-7{beta}, 8{alpha}-dihydroxy-9{alpha}, 10{alpha}-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene on mouse skin and in the newborn mouse

Ellagic acid, quercetin and robinetin were tested for theirability to antagonize the tumor-initiating activity of benzo[a]pyrene(B[a]P) and (±)-7ß, 8-dihydroxy-9, 10-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene(B[a]P 7,8-diol-9,10-epoxide-2), the ultimate carcinogenic metaboliteof benzo[a]pyrene. Ellagic acid, robinetin or quercetin (2500nmol) had no tumor-initiating activity on mouse skin, but thetopical application of 2500 nmol of ellagic acid 5 min beforea tumor-initiating dose of 200 nmol of B[a]P 7,8-diol-9,10-epoxide-2caused a 59–66% inhibition in the number of skin tumorsper mouse that were observed after 15–20 weeks of promotionwith 12-O-tetradecanoylphorbol-13-acetate. Similar treatmentwith 2500 nmol of robinetin or quercetin caused a statisticallyinsignificant 16–24% inhibition in the tumor-initiatingactivity of 200 nmol of B[a]P 7,8-diol-9,10-epoxide-2 applied5 min later. Treatment of mice with 2500 nmol of ellagic acid5 min before the application of 50 nmol of B[a]P inhibited themean number of skin tumors per mouse by 28–33% after 15–20weeks of promotion, but these decreases were not statisticallysignificant. Robinetin and quercetin had little or no effecton the tumor-initiating activity of B[a]P on mouse skin. Treatmentof preweanling mice with 1/7, 2/7 and 4/7 of the total doseof ellagic acid (300 nmol), robinetin (1400 nmol), myricetin(1400 nmol) or quercetin (1400 nmol) i.p. on their first, eighthand fifteenth day of life, respectively, did not cause the formationof tumors in animals that were killed 9–11 months later.Similar treatment of preweanling mice with the above doses ofthe phenolic compounds 10 min before the i.p. injection of atotal dose of 30 nmol of B[a]P 7,8-diol-9,10-epoxide-2 duringthe animal's first 15 days of life caused a 44–75% inhibitionin the number of diol-epoxide-induced pulmonary tumors per mouse.Similar treatment with these plant phenols had little or noeffect on B[a]P-induced pulmonary tumors.     

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.     

Tumorigenicity of racemic and optically pure bay region diol epoxides and other derivatives of the nitrogen heterocycle dibenz[a,h]acridine on mouse skin

CD-1 female mice were initiated with a single topical application of 500 nmol dibenz[a,h]acridine (DB[a,h]Acr), its racemic trans-1,2-, 3,4-, 8,9- and 10,11-dihydrodiols, racemic DB[a,h]Acr 3,4-diol 1,2-epoxide-1 and -2 or racemic DB[a,h]Acr 10,11-diol 8,9-epoxide-1 and -2, where the benzylic hydroxyl group is either cis (isomer 1) or trans (isomer 2) to the epoxide oxygen. The mice were subsequently treated twice weekly with 12-O-tetradecanoylphorbol 13-acetate for 25 weeks. High tumorigenicity was observed only for DB[a,h]Acr, its 10,11-dihydrodiol and DB[a,h]Acr 10,11-diol 8,9-epoxide-2 (3.3, 1.2 and 1.6 tumors/mouse, respectively). The tumor-initiating activity of a 50 nmol dose of DB[a,h]Acr and the optically active (+)- and (-)-enantiomers of DB[a,h]Acr 10,11-dihydrodiol and of the optically active DB[a,h]Acr 10,11-diol 8,9-epoxide-1 and -2 were also studied. Only DB[a,h]Acr, (-)-DB[a,h]Acr (10R,11R)-dihydrodiol and the bay region (+)-(8R,9S,10S,11R)-diol epoxide-2 were highly active (1.6, 1.7 and 2.4 tumors/mouse, respectively). These results are consistent with previous studies which showed that the corresponding bay region RSSR diol epoxides of benzo[a]pyrene, benz[a]anthracene, chrysene and benzo[c]phenanthrene as well as the aza-polycyclic dibenz[c,h]acridine are the most tumorigenic isomers.     

Synthesis of fjord region tetraols and their use in hepatic biotransformation studies of dihydrodiols of benzo[c]chrysene, benzo[g]chrysene and dibenzo[a,l]pyrene

Metabolic activation of the racemic benzo[c]chrysene-trans-9,10-, benzo[g]chrysene-trans-11,12- and dibenzo[a,l]pyrene-trans-11,12- dihydrodiols to fjord region syn- and anti-dihydrodiol epoxides by microsomes of Aroclor 1254-treated Sprague-Dawley rats has been examined. Since the fjord region dihydrodiol epoxides were hydrolytically unstable under the experimental conditions, their enzymatic formation was determined by analyzing the tetraols as their products of acidic hydrolysis upon addition of perchloric acid. The various stereoisomeric tetraols formed were separated by HPLC and identified by co-chromatography with authentic tetraols, which had been prepared by acidic hydrolysis of synthetically available syn- and anti- dihydrodiol epoxides and characterized by NMR and UV spectroscopy. Under standardized conditions the acidic hydrolysis of syn-dihydrodiol epoxides of benzo[c]chrysene, benzo[g]chrysene and dibenzo[a,l]pyrene resulted in the formation of two tetraols with cis/trans ratios of 81:19, 77:23 and 80:20, respectively, whereas the anti-dihydrodiol epoxides underwent almost exclusively trans hydrolysis. The proportion of the stereoisomeric tetraols obtained from microsomal incubations indicates that all three dihydrodiols are predominantly oxidized at the adjacent olefinic double bond to the anti-diastereomers of the corresponding fjord region dihydrodiol epoxides accounting for 4-35% of the ethyl acetate-extractable metabolites. To allow quantitative assessment of the metabolites 3H-labeled trans-dihydrodiols were synthesized by reduction of the corresponding o-quinones with sodium borotritide. Metabolic conversion of benzo[c]chrysene-trans-9,10- and dibenzo[a,l]pyrene-trans-11,12-dihydrodiol by rat liver microsomes were in a similar low range during the first 10 min of incubation (6.2 +/- 1.2 and 3.4 +/- 1.0 nmol substrate/nmol cytochrome P450/10 min, respectively), whereas the conversion of benzo[g]chrysene-trans-11,12- dihydrodiol was much higher (20.6 +/- 2.2 nmol substrate/nmol cytochrome P450/10 min). Given the strong intrinsic mutagenic and carcinogenic activity of the fjord region dihydrodiol epoxides, our data indicate that their formation, even at a relatively low level, may contribute significantly to the biological activity of the parent hydrocarbons.      

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.      

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.     

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.