Tumor-initiating activity of 4-fluoro-7,12-dimethylbenz[a]anthracene and 1,2,3,4-tetrahydro-7,12-dimethylbenz[a]anthracene in female SENCAR mice

We have determined the skin tumor-initiating activity in SENCARmice of two A-ring derivatives of 7,12-dimethylbenz[a]anthracene(DMBA). 4-Fluoro-7,12-dimethyibenz[a]-anthracene at a dose of200 nmol per mouse exhibited weak activity, producing 0.6 papillomasper mouse; doses of 10 and 20 nmol per mouse had no activity.A derivative of DMBA with the A-ring reduced, l,2,3,4-tetrahydro-7,12-dimethyl-benz[a]anthracene(1,2,3,4,-H₄-DMBA), had substantial tumor-initiating activitywhen compared with the parent hydrocarbon. In one experiment,doses of 10 and 100 nmol per mouse gave rise to 1.6 and 9.5papillomas per mouse, respectively; similar results were obtainedin 3 additional experiments. Although the tumor-initiating activityof 1,2,3,4,-H₄-DMBA was approximately one-tenth that of DMBA,this derivative was slightly (17%) more active than 3-methylcholanthreneand 3 times more active than benzo[a]pyrene. 1,2,3,4-H₄-DMBAwas tested for the ability to induce mutations to 6-thioguanine-resistancein Chinese hamster V79 cells. In the absence of feeder cellscapable of metabolizing polycyclic hydrocarbons, it was notmutagenic. However, in a cell-mediated mutation assay with secondaryhamster embryo cells as activators, this derivative producedmutations in a dose-dependent manner and was approximately one-tenthas active as DMBA. These results indicate that metabolism ofDMBA at positions 1-, 3-, 2- and 4- is important for biologicalactivity and that for certain derivatives (i.e., 1,2,3,4-H₄-DMBA),alternate pathways of metabolic activation may also be important.     

Correlation between formation of a specific hydrocarbon-deoxyribonucleoside adduct and tumor-initiating activity of 7,12-dimethylbenz(a)anthracene and its 9- and 10-monofluoroderivatives in mice

The formation of epidermal DNA adducts from 9-fluoro-7,12-dimethylbenz(a)anthracene (9-F-DMBA) was compared with 7,12-dimethylbenz(a)anthracene (DMBA) and 10-fluoro-7,12-dimethylbenz(a)anthracene (10-F-DMBA) in SENCAR mice. 9-F-DMBA is equipotent, whereas 10-F-DMBA is more potent than DMBA for skin tumor initiation in this mouse stock. The quantity of covalently bound DNA adducts was essentially identical between 9-F-DMBA and DMBA at all doses tested in the range of 10 to 100 nmol/mouse. These results correlated closely with the dose-response relationships for tumor initiation by the two hydrocarbons. A quantitative comparison of the hydrocarbon-DNA adducts formed after topical application of 100 nmol of DMBA, 9-F-DMBA, and 10-F-DMBA yielded interesting results. The total binding for the three hydrocarbons at this dose was 16.2 +/- 2.6, 18.4 +/- 2.4, and 52.3 +/- 6.8 pmol/mg of epidermal DNA, respectively. Analysis of these DNA adduct samples by dihydroboronate chromatography demonstrated marked reductions in the percentage of syn-diol-epoxide-DNA adducts with both 9-F-DMBA (24%) and 10-F-DMBA (18%) compared with DMBA (57%). Analysis of DNA adduct samples from DMBA-, 9-F-DMBA-, and 10-F-DMBA-treated mice (100 nmol/mouse) by high-pressure liquid chromatography revealed qualitatively similar profiles. However, a quantitative comparison of the three major DNA adducts, tentatively identified as anti-diol-epoxide-deoxyguanosine (Peak I), syn-diol-epoxide-deoxyadenosine (Peak II), and anti-diol-epoxide-deoxyadenosine (Peak III), revealed significant differences. With both 9-F-DMBA and 10-F-DMBA there were marked increases (236% and 644%, respectively) in the quantity of Peak I compared to DMBA. On the other hand, Peak II was formed in approximately equal amounts with DMBA and 10-F-DMBA but only 50% of the DMBA value with 9-F-DMBA. Interestingly, Peak III was formed in approximately equal amounts with both DMBA and 9-F-DMBA but was increased to 337% of the DMBA value with 10-F-DMBA. Thus, the actual level of Peak III (tentatively identified as anti-diol-epoxide-deoxyadenosine) correlated closely with the tumor-initiating activity of these three hydrocarbons, whereas the levels of the other two adducts did not. These data suggest that formation of a specific DNA adduct may be important for DMBA skin tumor initiation. These data are discussed in relation to skin tumor initiation by other hydrocarbons.     

Cell and microsome mediated binding of 7,12-dimethylbenz(a)anthracene to DNA studied by fluorescence spectroscopy.

Fluorescence spectra of DNA isolated from hamster embryo cells incubated with 7,12-dimethylbenz(a)anthracene, or DNA modified in a microsomal system by reaction with this carcinogen or its 7-hydroxymethyl derivative, were compared to various model compounds. The spectra indicate that the DMBA derivative bound to DNA, in all 3 cases, has a 9,10-dimethylanthracene-like chromophore. They also provide the first evidence of the similarity in structure of the DNA-bound products between 7,12-dimethylbenz(a)anthracene and its 7-hydroxymethyl derivative. Our results are consistent with an activation mechanism that involves saturation of the 1,2,3,4-ring positions.     

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.     

Kinetics of formation and disappearance of 7,12-dimethylbenz(a)anthracene:DNA adducts in mouse epidermis

The rates of formation and disappearance of 7,12-dimethylbenz(a)anthracene (DMBA):DNA adducts were analyzed in the epidermis of SENCAR mice over a 21-day time course. Mice were treated topically with 10 nmol of tritium-labeled DMBA per mouse at various times prior to sacrifice. Under these experimental conditions, total covalent binding of DMBA to epidermal DNA reached a peak at 24 h, and thereafter, DMBA:DNA adduct disappearance was biphasic. The early phase of DMBA:DNA adduct disappearance (Phase A) between 24 and 72 h had a half-life of 3.17 +/- 1.1 days, whereas the later phase (Phase B) had a half-life of 6.46 +/- 1.3 days. A comparison of the biphasic disappearance of total DMBA:DNA adducts with total benzo(a)pyrene:DNA adducts at comparable tumor-initiating doses (i.e., doses producing similar papilloma responses in SENCAR mice) revealed that the half-life for Phase A disappearance of benzo(a)pyrene:DNA adducts was approximately 3 times faster than for DMBA:DNA adducts (1.08 +/- 0.3 days versus 3.17 +/- 1.1 days), respectively. Phase B disappearance of DNA adducts was essentially identical for both hydrocarbons and was similar to the rate of loss of label in epidermal DNA due to cell turnover. The rates of formation and disappearance of the three major DNA adducts derived from DMBA were also examined. Peaks II (syn-diol-epoxide deoxyadenosine) and III (anti-diol-epoxide deoxyadenosine) disappeared more rapidly than Peak I (anti-diol-epoxide deoxyguanosine) beyond 24 h. The data support the conclusion that, for a particular hydrocarbon such as DMBA, deoxyadenosine adducts disappear from epidermal DNA faster than the corresponding deoxyguanosine adducts. In addition, the data suggest that, at the doses used, total DMBA:DNA adducts disappear initially more slowly from epidermal DNA than benzo(a)pyrene:DNA adducts.     

Tumorigenesis of mammary gland by 7,12-dimethylbenz(a)anthracene during pregnancy: relationship with DNA synthesis

The relationship between mammary cell proliferation during pregnancy and susceptibility to 7,12-dimethylbenz(a)anthracene (DMBA) was examined. DMBA was administered intravenously to Sprague-Dawley rats on the 5th, 10th or 15th day of pregnancy. [3H]thymidine labelling index (LI) of the mammary cells at the time of treatment with the carcinogen was determined and found to be higher in the pregnant rats than in age-matched virgin controls. In spite of the high proliferative index of the mammary cells, significant inhibition of tumorigenesis occurred in the pregnancy rats allowed to complete pregnancy and parturition following treatment with DMBA. However, when pregnancy was terminated by cesarian section shortly after treatment with DMBA, there was a significantly higher tumor incidence as compared to the "full-term" rats. It was observed that the earlier the pregnancy was terminated, the greater was the incidence of mammary tumors. This would indicate that the inhibitory effect of pregnancy is related to changes occurring during the later half of gestation. The differentiation of mammary cells for milk synthesis as pregnancy progresses is postulated to be a major reason for the observed refractoriness of the mammary cells to DMBA at that time.     

Age-related modification of 7,12-dimethylbenz[a]anthracene binding to rat mammary gland DNA.

The binding of 7,12-dimethylbenz[a]anthracene (DMBA) to mammary gland and liver DNA of female Sprague-Dawley rats either 35, 50, or 120 days of age at the time of carcinogen administration was studied. Following a single oral feeding of tritium-labeled DMBA, the level of binding to liver DNA of rats in all 3 age groups was significantly lower, at all times during a 6-week period, than that of binding to mammary DNA. The amount of DMBA bound to liver DNA was a function of the amount of carcinogen administered and not the age of the animal. In contrast, DMBA binding to mammary DNA was dependent on the age of the animal at the time of carcinogen feeding. Furthermore, in the age group with 100% tumor induction (50 days old), DMBA binding increased directly with the amount of carcinogen fed; this was not the case for the other 2 age groups. These results indicated that a significant correlation existed between the age of the rat, the amount of DMBA bound to DNA, and the incidence of mammary tumors following carcinogen feeding.     

Benzo(a)pyrene and 7,12-dimethylbenz(a)anthracene metabolism and DNA adduct formation in primary cultures of hamster epidermal cells

Primary cultures of hamster epidermal cells exposed to hydrocarbon, 1 microgram/ml, rapidly metabolized [3H]benzo(a)pyrene and [14C]7,12-dimethylbenz(a)anthracene to ethyl acetate:acetone- and water-soluble metabolites. By 24 hr, only 13.6% of the organic solvent-soluble radioactivity recovered in the medium was unchanged [3H]benzo(a)pyrene, and only 5.9% was unchanged [14C]7,12-dimethylbenz(a)anthracene. With both hydrocarbons, the major water-soluble metabolites found extracellularly were conjugated with glucuronic acid; these were primarily phenolic derivatives. Metabolites cochromatographing with 7,8-dihydro-7,8-dihydroxybenzo(a)pyrene or trans-3,4-dihydro-3,4-dihydroxy-7,12-dimethylbenz(a)anthracene were not detectable in high-pressure liquid chromatographic profiles of organic solvent-soluble intracellular and extracellular metabolites. However, analysis of [3H]benzo(a)pyrene: and [3H]7,12-dimethylbenz(a)anthracene: DNA adducts indicated that these putative proximate carcinogenic metabolites were formed in these cells and subsequently metabolized to DNA-binding products. The results suggest that metabolic incompetence may not be an explanation for the relative resistance of the hamster to epidermal carcinogenesis by polycyclic hydrocarbons.     

Food restriction inhibits [3H] 7,12-dimethylbenz(a)anthracene binding to mouse skin DNA and tetradecanoylphorbol-13-acetate stimulation of epidermal [3H] thymidine incorporation

It has been known for many years that reducing the food intake of laboratory mice and rats inhibits the development of a broad spectrum of chemically induced and spontaneous tumors, but the mechanism of this effect is poorly understood. Food restriction of A/J mice for two weeks is now shown to inhibit the binding of topically applied [3H]7,12-dimethylbenz(a)anthracene (DMBA) to skin DNA by 50% and to abolish the stimulation of [3H]-thymidine incorporation in the epidermis produced by topical application of the tumor promoter tetradecanoylphorbol-13-acetate (TPA). Similar effects on the actions of DMBA and TPA are observed following topical application of the adrenal steroid, dehydroepiandrosterone (DHEA), a potent glucose-6-phosphate dehydrogenase (G6PDH) inhibitor, while food restriction for two weeks depresses epidermal G6PDH activity by 60%. It is suggested that both the inhibition of [3H]DMBA binding to skin DNA and the TPA stimulation in epidermal [3H]thymidine incorporation result from a reduction in the NADPH cellular pool as a result of G6PDH inhibition.     

Influence of the rate of 7,12-dimethylbenz[a]anthracene metabolic activation, in vivo, on its binding to epidermal DNA and skin carcinogenesis

The effects of skin pretreatment of two strains of Swiss mice,NMRI and CF₁, with ß-naphthoflavone on epidermal tumorsinduced by 7,12-dimethylbenz[a]anthracene (DMBA) have been studied.NMRI Swiss strain exhibits a natural high sensitivity to thecarcinogenic effect of the hydrocarbon, and the pretreatmentof mice by ß-naphthoflavone leads to a large decreaseof the formation of tumors. In contrast, CF₁ Swiss mice exhibita very low sensitivity to the carcinogenic effect of DMBA anda dramatic increase of the tumorigenicity occurs when the animalsare pretreated by the inducer. Aryl hydrocarbon hydrox-ylaseactivity in skin homogenates of control and differently inducedNMRI and CF₁ mice has been measured and compared to (a) thekinetics of disappearance of [³H]DMBA from skin in control andß-naphthoflavone-treated mice and (b) the kineticsof the covalent binding of the radioactive hydrocarbon to skinepidermal DNA (in control and ß-naphthoflavone-treatedmice). The comparison between these three parameters and thetumorigenicity experiments seem to indicate that an optimalrate of DMBA metabolic activation exists which corresponds toa high level of skin tumors correlated with DNA binding of thepolycyclic hydrocarbon. These data might explain some conflictingresults which have been reported on the effect of in-ducersof aryl hydrocarbon hydroxylases on tumor induction by polycyclicaromatic hydrocarbons.     

A beef-derived mutagenesis modulator inhibits initiation of mouse epidermal tumors by 7,12-dimethylbenz[a]anthracene

Ground beef contains an organic solvent extractable mutagenesismodulator. Crude or partially-purified preparations of the activitywere applied to the backs of SENCAR or CD-1 mice 5 min beforeapplication of 7,12-dimethylbenz[a]anthracene (DMBA). Controlsreceived solvent (acetone) only prior to DMBA. Following 1–2week intervals promotion was effected with twice-weekly applicationsof 12-O-tetradecanoylphorbol-13-acetate. Modulator-treated miceconsistently developed fewer papillomas and exhibited lowertumor incidences than did positive control mice.     

Evidence that binding of 7,12-dimethylbenz(a)anthracene to DNA in mouse embryo cell cultures results in extensive substitution of both adenine and guanine residues

Primary mouse embryo cell cultures were grown in the presence of [14C]guanine, labeling primarily deoxyguanosine residues in the cellular DNA, or in the presence of [14C]adenine, labeling both deoxyguanosine and deoxyadenosine residues in the cellular DNA. These cultures were subsequently exposed to 7,12-[3H]dimethylbenz(a)anthracene for 24 hr. The DNA was isolated and hydrolyzed to deoxyribonucleosides, and the 7,12-dimethylbenz(a)anthracene:deoxyribonucleoside adducts were separated chromatographically allowing the three major adducts found to be identified as bay-region anti-dihydrodiol-epoxide:deoxyguanosine and :deoxyadenosine adducts and a bay-region syn-dihydrodiol-epoxide:deoxyadenosine adduct. Therefore, in contrast to what is known for benzo(a)pyrene, substantial amounts of deoxyadenosine adducts are formed with the more potent carcinogen, 7,12-dimethylbenz(a)anthracene.     

Comparative metabolism and DNA binding of 7,12-dimethylbenz[a]anthracene and its weakly carcinogenic 5-fluoro analog.

Cultured Syrian hamster embryo cells readily convert both the carcinogenic polycyclic aromatic hydrocarbon, 7,12-dimethylbenz[a]anthracene (DMBA), and its weakly carcinogenic analogue, 5-fluoro-7,12-dimethylbenz[a]anthracene (5F-DMBA), to water soluble metabolites. DMBA, however, binds to the hamster cell DNA at least 2.8-3.0 times more extensively than 5F-DMBA. Likewise, 5F-DMBA is converted to water soluble metabolites by liver microsomal preparations at a rate equal to DMBA, but the latter compound binds to DNA 2.6-3.2 times more effectively.     

Photosensitivity of DNA-bound 7,12-dimethylbenz(alpha)anthracene.

Structural information about the products formed when 7,12-dimethylbenz(alpha)anthracene (DMBA) is bound to DNA in mammalian cell cultures has been sought through studies of the photosensitivities of these products and of various model compounds. Under conditions of light exposure in which the DNA-DMBA products were highly photosensitive, 8,9,10,11-tetrahydro-DMBA and 5,6-dihydro-DMBA were stable, whereas 9,10-dimethyl-anthracene and DMBA itself were highly photosensitive. This indicates that in the binding reaction with DNA, DMBA retains either the aromatic benz(alpha)anthracene nucleus or is metabolically activated in the 1,2,3,4-ring.