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.     

Biosynthesis of the potent carcinogen 7,12-dimethylbenz[a]anthracene

Earlier studies from this laboratory of the metabolism of 7,12-dimethylbenz[a]anthracene (DMBA) and of benzo[a]pyrene as well as studies of mutagenic and carcinogenic activity of some of the metabolic products led to the concept that a necessary first step in carcinogenesis by most alkyl substituted polycyclic hydrocarbons is biotransformation to a meso-anthracenic hydroxyalkyl metabolite, whereas most hydrocarbons lacking alkyl substituents undergo a bio-alkylation substitution reaction in the mesoanthracenic position(s) or L-region as a necessary first step in carcinogenesis. According to this unified hypothesis, all strong polycyclic hydrocarbon carcinogens must either, themselves, bear a meso-anthracenic alkyl substituent or else undergo a bio-alkylation substitution reaction in vitro and in vivo. Here we report that the weak carcinogen benz[a]anthracene undergoes meso-anthracenic methylation by S-adenosyl-L-methionine (SAM), in the presence of a rat liver cytosol preparation, in vitro, to form DMBA and presumably the moderately active carcinogens 7-methylbenz[a]anthracene and 12-methylbenz[a]anthracene. These compounds are substrates for further L-region methylation to form the strong carcinogen, DMBA.     

In vitro interaction of 7,12-dimethylbenz[a]anthracene and its biliary metabolites with dietary fiber

This in vitro study examined the ability of various dietary fiber components to bind 7,12-dimethylbenz[a]anthracene (DMBA), a hydrocarbon carcinogen known to contaminate food. Bile salt solutions of DMBA were incubated with individual fiber materials under various conditions, and the degree of binding was assessed. Binding of DMBA from simple bile salt solution was greatest with delipidated bran greater than acid lignin greater than alpha-cellulose greater than unprocessed bran. Binding to acid lignin was slowly reversible, was constant within the pH range 4-8, and appeared independent of a bile salt-fiber interaction. Acid lignin bound DMBA from a mixed lipid-bile salt solution significantly, but less effectively than from pure bile salt solutions. DMBA biliary metabolites from Sprague-Dawley rats were bound less extensively to all the fiber materials than was the parent hydrocarbon, but following hydrolysis of the metabolites by beta-glucuronidase binding was significantly increased. These results indicate that there is considerable potential for dietary fiber to interact with DMBA and its metabolites in the lumen of the gastrointestinal tract, possibly influencing hydrocarbon carcinogen bioavailability.     

Light-dependent activation of 7,12-dimethylbenz[a]anthracene to a potent genotoxicant

7, 12-Dimethylbenz[a]anthracene (DMBA), which is widely usedin mutagenesis and experimental carcinogenesis, is activatedto a mutagen by white fluorescent light. A 40 min exposure towhite fluorescent light of Salmonella typhimurium TA98 platestreated with DMBA, in the absence of exogenous metabolism, resultedin an 30-fold increase in the number of histidine revertants.This phenomenon also occurs, with lesser intensity, with otherpromutagens, such as benzo[a]pyrene or 2-acetylamino-fluorene,and in other Salmonella tester strains. Moreover, white fluorescentlight is able to activate DMBA to a toxicant for Chinese hamsterV79 cells in culture, resulting in very low cell survival. Underthese conditions, white fluorescent light-activated DMBA wasshown to cause chromosomal aberrations, but not gene mutations,as determined by resistance to thioguanine. This white fluorescentlight-dependent activation of DMBA seems to be related to theformation of reactive species, as the addition of vitamin Eresults in a reduction in the number of histidine revertantsinduced by white fluorescent light in S.typhimurium TA98.     

Prior dietary protein intake and DNA-binding 7,12-dimethylbenz[a]anthracene metabolites formed by isolated rat hepatocytes

Hepatocytes were isolated from noninbred Sprague-Dawley rats previously fed diets containing 7.5 or 15% protein. These hepatocytes were incubated in the presence of exogenous DNA for examination of their ability to metabolize 7,12-dimethylbenz[a]anthracene [(DMBA) CAS: 57-97-6] and release reactive DNA-binding metabolites to the medium. An increased formation of extracellular water-soluble metabolites of DMBA was observed in hepatocyte cultures from rats fed a 15% protein diet compared with that in cells from rats fed 7.5% protein. As dietary protein increased, there was a reduction in the release of DNA-binding metabolites by isolated hepatocytes. Bay-region dihydrodiol-epoxide adducts were formed with extracellular calf thymus DNA and hepatic DNA. However, most of the binding of DMBA with extracellular and intracellular DNA was due to unidentified DMBA-DNA adducts that eluted, upon reversed-phase chromatography, after the bay-region dihydrodiol-epoxide DMBA adducts were formed. The present studies show that feeding animals diets that are limiting in protein results in a decrease in DMBA detoxification and an increase in excretion of reactive DMBA metabolites from the liver. These results may explain the previously observed influence of dietary protein on the initiation of DMBA-induced mammary carcinogenesis in the rat.     

Oxidative metabolism of 7-methylbenz[a]anthracene, 12-methylbenz[a]anthracene and 7,12-dimethylbenz[a]anthracene by rat liver cytosol

Earlier studies from this laboratory demonstrated that benz[a]anthracene (BA), 7-methylbenz[a]anthracene (7-MBA) and 12-methylbenz[a]anthracene (12-MBA) undergo a bio-alkylation substitution reaction in the meso-anthracenic position(s) or L-region leading to the biosynthesis of the potent carcinogen 7,12-dimethylbenz[a]anthracene (7,12-DMBA). These results support the hypothesis that for most, if not all, unsubstituted polycyclic aromatic hydrocarbon carcinogens, the chemical or biochemical introduction of an alkyl group in the meso-anthracenic position(s) or L-region is a structural requirement for strong carcinogenic activity. Here we report that the L-region methyl derivatives 7-MBA, 12-MBA and 7,12-DMBA are oxidized to hydroxymethyl derivatives by a rat liver cytosol preparation without any apparent oxidation of the ring positions.     

Molecular structure of the K-region cis-dihydrodiol of 7,12-dimethylbenz[a]anthracene.

The molecular structure and conformation of the cis-5,6-dihydrodiol of 7,12-dimethylbenz[a]anthracene has been determined by an X-ray crystallographic analysis. The compound crystallizes in the space group P21/a with cell dimensions a equals 17.799(6), b equals 33.211(8), c equals 5.241(1) A, beta equals 91.88(2)degrees. There are two molecules, designated A and B in the asymmetrical unit, that are not related to each other by crystallographic symmetry. Their conformations are almost identical, and there are no significant differences in their bond lengths or angles. In both molecules the 5-hydroxyl group is equatorial while the 6-hydroxyl group is axial. This conformation is probably forced by steric hindrance between the hydroxyl group, 0-6, and the hydrogen atoms of the 7-methyl group. The molecules pack in the crystal by forming hydrogen bonds between the hydroxyl groups of adjacent molecules, A with A, B, with B, and A with B. The ring system of the cis-5,6-dihydrodiol is much more buckled than is that in 7,12-dimethylbenz[a]anthracene itself. The angle between the two outermost rings is 36 degrees, the deviation from planarity being primarily a consequence of the partial saturation in the ring containing the two hydroxyl groups. Extrapolation of these results to other dihydrodiol derivatives of carcinogenic hydrocarbons permits some predictions of preferred molecular geometry. Thus, the 8,9-dihydrodiol-10,11-epoxide of 7,12-dimethylbenz]a[anthracene, analogous to the biologically active 7,8-dihydrodiol-9,10-epoxide of benzo]a[pyrene, a mutagen that is believed to be an active intermediate in carcinogenesis by benzo]a[pyrene, should probably exist preferentially in a conformation bearing the8-hydroxyl group in the axial orientation.     

Comparison of mutagenesis and malignant transformation by dihydrodiols from benz[a]anthracene and 7,12-dimethylbenz[a]anthracene.

Five dihydrodiols derived from benz[a]anthracene (BA) and 4 dihydrodiols derived from 7,12-dimethylbenz[a]anthracene (DMBA) have been tested, together with the parent hydrocarbons, for their abilities to induce mutations to 8-azaguanine resistance in V79 (Chinese hamster cells and malignant transformation in M2 mouse fibroblasts. The syn- and anti-isomers of benz[a]anthracene 8,9-diol 10,11-oxide were also tested for biological activity in these two systems. The non-K-region 1,2- and 3,4-dihydrodiols of BA induced mutations but the non-K-region 8,9-dihydrodiol and the K-region 5,6-dihydrodiol were inactive as mutagens; none of these BA diols transformed M2 mouse fibroblasts. The 3,4- and the 8,9-dihydrodiols derived from 7,12-dimethylbenz[a]anthracene induced mutations in V79 cells and malignant transformation in M2 mouse fibroblasts and both were more active than the hydrocarbon itself. The K-region 5,6-dihydrodiol and the non-K-region 10,11-dihydrodiol of DMBA were inactive in both test systems. The results are not inconsistent with other data suggesting that the metabolic activation of both BA and DMBA occurs through conversion of the respective 3,4-dihydrodiols into the related vicinal diol-epoxides, although other dihydrodiols may also be involved in vivo. Both the BA diol-epoxides tested were mutagenic, but although the anti-isomer transformed M2 fibroblasts, the syn-isomer was inactive.     

Estrogenic properties of 3,9-dihydroxy-7,12-dimethylbenz[a]anthracene in rats.

Previously, the 3,9-dihydroxy derivative of benz[a]-anthracene was shown to be weakly estrogenic. The availability of the related diol of the mammary carcinogen dimethylbenz[a]-anthracene, i.e., 3,9-dihydroxy-7,12-dimethylbenz[a]anthracene (3,9-diOHDMBA), prompted a similar study of its estrogenic properties. The competitive binding studies of 3,9-diOHDMBA with 17beta-estradiol in the uterine cytosol of immature SD rats gave a Ka of 1.7 x 10(8) M-1. 17beta-Estradiol (10(-9) M) binding to the 8S binding protein was inhibited by 3,9-diOHDMBA at concentrations similar to those of nafoxidine HCl (1 x 10(-5) M). Bioassay demonstrated that the diol possesses 1/4,464 the activity of 17beta-estradiol.     

Effect of 7,12-dimethylbenz[a]anthracene on production and action of gamma interferon

Spleens were removed from Swiss Webster mice, and cultures of spleen cells were prepared. Gamma (ty pe II immune) interferon (IFN) production was induced in these cells by addition of the purified protein form of phytohemagglutinin (PHA-P) to the cultures. When 7,12-dimethylbenz[a]anthracene (DMBA) was added to the cultures either before or after addition of PHA-P, the production of gamma IFN was inhibited. The degree of inhibition was greater when the cells were treated with DMBA before addition of PHA-P. In experiments to determine the effects of DMBA on the antivirus activity of previously prepared gamma IFN preparations, DMBA was either added to target cells before the addition of exogenous gamma IFN or mixed together with exogenous gamma IFN. In both cases. the antivirus activity of this exogenous gamma IFN was not affected. These data suggest that DMBA treatment can inhibit the production but not the antivirus activity of murine gamma IFN.     

Effect of nicotine on 7,12-dimethylbenz[a]anthracene carcinogenesis in hamster cheek pouch

We divided 40 male Syrian golden hamsters into four groups of 10 animals each, and we treated both cheek pouches of each hamster three times a week as follows: group 1, 50 microL of sesame oil; group 2, 50 microL of 6% nicotine in sesame oil; group 3, 50 microL of 1% 7,12-dimethylbenz[a]anthracene (DMBA) in sesame oil; and group 4, 50 microL of 1% DMBA in 6% nicotine in sesame oil. Cheek pouches were examined clinically and histologically after 12 weeks of treatment. Hamsters treated with DMBA and nicotine showed significantly (P less than .001) more tumors and a significantly (P less than .05) greater-than-expected proportion of large tumors (greater than or equal to 3-mm diameter) than hamsters treated with DMBA alone. Histologically, there was a greater degree of dysplasia in lesions from the group receiving DMBA plus nicotine than in the DMBA only group. The results suggest that nicotine acts as a cofactor in DMBA tumorigenesis.     

Formation of the 1,2-diol as a metabolite of 7,12-dimethylbenz[a]anthracene by rodent and human skin

Rodent and human skin maintained in short-term organ culturewas treated with ³H-labelled 7,12-dimethylbenz[a]anthracene.Extracts of the rodent tissue and culture fluid in which eithermouse, rat or human skin had been maintained were found to containradioactive material that possessed the chromatographic characteristicsof trans-1,2-dihydro-1,2-dihydroxy-7, 12-dimethylbenz[a]anthracenewhen it was examined in two different h.p.l.c. systems. Whenthe metabolite was treated with hot mineral acid, the two radioactiveproducts formed co-chromatographed with the phenols that wereformed when the reference dihydrodiol was similarly treated.Acetylation of the isolated metabolite yielded a single productthat had chromatographic properties identical to those of thediacetate of the reference dihydrodiol. Taken together thesedata show that the 1,2-dihydrodiol of 7,12-dimethylbenz[a]anthraceneis formed as a metabolite of this hydrocarbon by rodent andhuman skin maintained in short-term organ culture.     

Chromatographic and fluorescence spectroscopic studies of individual 7,12-dimethylbenz[a]anthracene - deoxyribonucleoside adducts

Compared with standard Sephadex LH-20 column chromatography,a newly developed high pressure liquid chromatographic separationof hydrocarbon deoxyribonucleoside adducts derived from theDNA of mouse embryo cell cultures exposed to 7,12-dimethylbenz[a]anthracene(DMBA) provides markedly superior resolution. Once resolved,the fluorescence spectroscopic properties of the three majorDMBA–DNA adducts indicate that the fluorescence exhibitedby adducts derived from a bay region syn dihydrodiol epoxideof DMBA differs subtly from that exhibited by adducts derivedfrom the isomeric anti dihydrodiol epoxide.     

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.     

Inhibitory effects of curcumin on tumor initiation by benzo[a]pyrene and 7,12-dimethylbenz[a]anthracene.

The effects of topical administration of curcumin on the formation of benzo[a]pyrene (B[a]P)-DNA adducts and the tumorigenic activities of B[a]P and 7,12-dimethylbenz[a]anthracene (DMBA) in epidermis were evaluated in female CD-1 mice. Topical application of 3 or 10 mumol curcumin 5 min prior to the application of 20 nmol [3H]B[a]P inhibited the formation of [3H]B[a]P-DNA adducts in epidermis by 39 or 61% respectively. In a two-stage skin tumorigenesis model, topical application of 20 nmol B[a]P to the backs of mice once weekly for 10 weeks followed a week later by promotion with 15 nmol 12-O-tetradecanoylphorbol-13-acetate (TPA) twice weekly for 21 weeks resulted in the formation of 7.1 skin tumors per mouse, and 100% of the mice had tumors. In a parallel group of mice, in which the animals were treated with 3 or 10 mumol curcumin 5 min prior to each application of B[a]P, the number of tumors per mouse was decreased by 58 or 62% respectively. The percentage of tumor-bearing mice was decreased by 18-25%. In an additional study, topical application of 3 or 10 mumol curcumin 5 min prior to each application of 2 nmol DMBA once weekly for 10 weeks followed a week later by promotion with 15 nmol TPA twice weekly for 15 weeks decreased the number of tumors per mouse by 37 or 41% respectively.     

Ras gene mutations in 7,12-dimethylbenz[a]anthracene (DMBA)-induced rat sarcomas

Tumor induction in rats by 7,12-dimethylbenz[a]anthracene (DMBA) will generate malignancies that display reproducible chromosomal abnormalities involving rat chromosome (RNO) 2. Thus, it has been reported that rat DMBA erythroleukemias display RNO2 abnormalities, which in this case were closely correlated to mutations in the Nras oncogene located in RNO2q34. Our cytogenetic analysis in a series of 17 DMBA-induced rat sarcomas showed that 11 (65%) tumors had a significant increase in RNO2 copy number. Furthermore, the incidence of point mutations in codons 12, 13 and 61 of Hras, Kras, and Nras was examined in the same set of sarcomas, and mutations were detected in three (18%) tumors, in codon 61 of Kras (CAA-->CAT) (1 of 17) and Nras (CAA-->CTA) (2 of 17). We conclude that the high frequency of RNO2 gain was in accordance with previous studies of DMBA-induced rat neoplasms, supporting the idea of a significant role of RNO2 in DMBA carcinogenesis. However, there was no clear-cut relationship between activated Nras and gain of RNO2 material, implying that mutational activation of Nras is not the causative factor underlying the gain of RNO2 copy number in rat DMBA sarcomas, in contrast to what has been suggested for DMBA-induced erythroleukemias.