Differential excision of carcinogenic hydrocarbon-DNA adducts in mouse embryo cell cultures.

Primary mouse embryo cell cultures efficiently excise DNA damage introduced by the carcinogens 7-bromomethylbenz[$\text{a}$]anthracene and 3-methylcholanthrene but are inefficient in excision of damage introduced by 7,12-dimethylbenz[a]anthracene. Since exposure of the cells to the latter compound does not impair their capacity for excision of adducts introduced by the bromocompound, it is concluded the 7,12-dimethylbenz[$\text{a}$]anthracene-DNA adducts are intrinsically difficult to excise.     

Metabolism of benzo[a]pyrene and 7,12-dimethylbenz[a]anthracene in cultured human fetal aortic smooth muscle cells.

Cultured human fetal aortic smooth muscle cells derived from the abdominal aorta converted benzo[a]pyrene (BaP) and 7,12-dimethylbenz[a]anthracene (DMBA) $\text{via}$ cytochrome P-450-dependent monooxygenation to metabolites detectable by both a highly sensitive radiometric assay and high pressure liquid chromatography (HPLC). Cells incubated with ³H-BaP transformed this substrate primarily to phenols. ¹⁴C-DMBA was converted to metabolites that cochromatographed with 12-hydroxymethyl-7-methylbenz[a]anthracene, 7-hydroxymethyl-12-methylbenz-[a]anthracene, 7,12-dihydroxymethylbenz[a]anthracene, and trans-8,9-dihydrodiol-7,12-DMBA. Exposure of cells in culture to 13 μM 1,2-benz[a]anthracene resulted in increased oxidative metabolism of both BaP and DMBA. In the case of BaP, total phenol formation was increased, while with DMBA all metabilities detected by HPLC were increased. Support for the potential role of metabolism of polycyclic aromatic hydrocarbons by aortic smooth muscle cells in the etiology of atherosclerosis was obtained.     

Metabolism of 7,12-dimethylbenz[a]anthracene and its methyl-hydroxylated metabolites: formation of phenolic metabolites at the 2-positions.

The 1- and 2-positions of 7,12-dimethylbenz[a]anthracene (DMBA) were thought not to be involved in biotransformation to 1,2-epoxide and 1,2-dihydrodiol because of steric hindrance from the 12-methyl group (Biochem. Biophys. Res. Commun. $\text{85}$: 357–362, 1978). However, we have identified four 2-phenols as rat liver microsomal metabolites of DMBA and its methyl-hydroxylated metabolites, 7-hydroxymethyl-12-methylbenz[a]anthracene, 7-methyl-12-hydroxymethylbenz[a]-anthracene, and 7,12-dihydroxymethylbenz[a]anthracene. Our findings suggest that neither the 12-methyl group nor the 12-hydroxymethyl group blocks the microsomal oxygenations of the 1,2 positions of DMBA or its methyl-hydroxylated derivatives. The 2-phenols may be formed as nonenzymatic rearrangement products of the 1,2-epoxide intermediates, although their formations by a direct hydroxylation mechanism cannot be ruled out.     

Detection of aryl hydrocarbon hydroxylase activity in normal and neoplastic human breast epithelium

Studies were conducted to determine whether normal and/or neo-plastic (MCF-7) human breast epithelial cells contain the microsomal aryl hydrocarbon hydroxylase (AHH) which catalyses the conversion of polycyclic aromatic hydrocarbons (PAH) to carcinogenic intermediates. Low constitutive levels of AHH activity were found in homogenates of both normal human breast epithelial and MCF-7 cells. The addition of 7,12-dimethylbenz(a)anthracene (DMBA) to the culture medium of either cell type significantly increased AHH activity. Peak induction of hydroxylase activity occurred following the $\text{in}$$\text{vitro}$ addition of 10 μM DMBA. A time course of DMBA-induced AHH activity in both normal human breast epithelium and MCF-7 cells revealed maximal induction 16 hr after 10 μM DMBA was added to the culture medium. Benzo(a)pyrene (BP), 3-methylcholanthrene (MCA) and benz(a)anthracene (BA) also induced AHH activity in normal and MCF-7 cells. For example, the addition of 10 μM BP to the culture medium of either normal human breast epithelial or MCF-7 cells for 16 hr increased AHH activity 13.8 and 65.3-fold, respectively. For all PAH, the magnitude of AHH induction was substantially greater in MCF-7 than normal breast epithelial cells. Finally, α-naphthoflavone inhibited BA-induced AHH activity in MCF-7 cells. The study demonstrates the presence of a PAH-inducible AHH enzyme(s) in normal human breast epithelial cells grown in primary culture and in the human breast tumor cell line, MCF-7.     

Differences between products of binding of 7,12-dimethylbenz[a]anthracene to DNA in mouse skin and in a rat liver microsomal system.

Hydrocarbon-deoxyribonucleoside products from the DNA of mouse skin exposed $\text{in vivo}$ to 7,12-dimethylbenz[a]anthracene are chromatographically the same as the products formed in mouse embryo cell cultures. These products, which are known to arise through the generation of a diol-epoxide in the 1,2,3,4-ring of the hydrocarbon, are chromatographically separable from products that result from reaction of the K-region oxide of this hydrocarbon with DNA. However, when 7,12-dimethylbenz[a]anthracene is bound to DNA in the presence of a microsomal system analogous to those used in various carcinogen detection systems, the hydrocarbon-deoxyribonucleoside products co-chromatograph with the K-region oxide products. Differences in the profiles of metabolites formed in mouse embryo cell cultures and rat liver microsomal systems are consistent with the differences between the DNA-bound products in these two systems.     

Increase of cyclic AMP-dependent protein kinase type II as an early event in hormone-dependent mammary tumor regression.

Primary, 7,12-dimethylbenz(α)anthracene (DMBA)-induced mammary carcinoma in the rat contains cyclic adenosine 3′,5′-monophosphate (cAMP)-dependent and -independent forms of protein kinase. When growth of DMBA-induced tumors was arrested by either ovariectomy or N⁶,O²′-dibutyryl cAMP treatment of the host, the activity of cAMP-dependent protein kinase type II markedly increased in the tumor cytosol, as shown by DEAE-cellulose chromatography and autophosphorylation. The increase in activity of cAMP-dependent protein kinase was also demonstrable in the tumor cytosol and nuclei following $\text{in}$$\text{vitro}$ incubation of tumor slices with cAMP. These results suggest that protein kinase type II is involved in the regression of hormone-dependent mammary tumors.     

Analysis of the epoxide of 7,12-dimethylbenz(a)anthracene and its hydrolysis products by gas chromatography

Column chromatography and electron-capture gas chromatography have been applied to the separation and quantitative analysis of the K-region epoxide of 7,12-dimethylbenz(a)anthracene, 5-hydroxy-7,12-dimethylbenz(a)anthracene, and trans-5,6-dihydro-5,6-dihydroxy-7,12-dimethylbenz(a)anthracene. Deactivation of chromatographic alumina with water permitted quantitative elution of the three compounds. In a solution of acetone and water, the epoxide was hydrolyzed to the 5,6-diol, but none of the 5-hydroxy derivative was formed.     

Argininosuccinate synthetase as a marker of transformed Syrian hamster epidermal cells

Thirteen continuous lines of Syrian hamster epidermal cells were isolated following direct treatment of epidermal cells with N-methyl-N′-nitro-N-nitrosoguanidine, or from epidermal cells cultured from animals treated transplacentally with 7,12-dimethylbenz(a)anthracene or dimethylnitrosamine. The epidermal origin of the lines was evidenced by the presence of desmosomes with radiating tonofilaments, keratin fibers, and the ability of the cells to use citrulline in place of arginine corresponding with their high argininosuccinate synthetase activity.     

Bioactivation of procarcinogens to mutagens in human fetal and placental tissues.

Enzyme systems in homogenates of human fetal hepatic, pulmonary, adrenal, renal and placental tissues catalyzed the conversion of benzo(a)pyrene, 7, 12-dimethylbenz(a)anthracene and N-2-fluorenyl-acetamide to intermediary metabolites that produced mutations in $\text{Salmonella typhimurium}$. The mutagenic metabolites generated from the three procarcinogen/promutagens displayed specificity with respect to the auxotrophic strains, chemicals and tissues investigated.     

Microsome-mediated mutagenicities of the dihydrodiols of 7,12-dimethylbenz[a]anthracene: high mutagenic activity of the 3,4-dihydrodiol.

7,12-Dimethylbenz[a]anthracene and its 3,4-, 5,6-, 8,9- and 10,11-dihydrodiols have been tested for mutagenicity towards $\text{S}$. $\text{typhimurium}$ TA100 in the presence of rat-liver post-mitochondrial supernatants from Aroclor-treated rats. At non-toxic concentrations, the non-K-region 3,4-dihydrodiol was six-fold more active than the parent hydrocarbon. At these concentrations, the 8,9-dihydrodiol showed some mutagenic activity, but the 5,6- and 10,11-dihydrodiols were inactive.     

The formation of dihydrodiols by the chemical or enzymic oxidation of benz[a] anthracene and 7,12-dimethylbenz[a] anthracene.

When benz[a] anthracene was oxidised in a reaction mixture containing ascorbic acid, ferrous sulphate and EDTA, the non-K-region dihydrodiols, trans-1,2-dihydro-1,2-dihydroxybenz[a] anthracene and trans-3,4-dihydro-3,4-dihydroxybenz[a] anthracene together with small amounts of the 8,9- and 10,11-dihydrodiols were formed. When oxidised in a similar system, 7,12-dimethylbenz[a] anthracene yielded the K-region dihydrodiol, trans-5,6-dihydro-5,6-dihydroxy-7,12-dimethylbenz[a] anthracene and the non-K-region dihydrodiols, trans-3,4-dihydro-3,4-dihydroxy-7,12-dimethylbenz[a] anthracene, trans-8,9-dihydro-8,9-dihydroxy-7,12-dimethylbenz[a] anthracene, trans-10,11-dihydro-10,11-dihydroxy-7,12-dimethylbenz[a] anthracene and a trace of the 1,2-dihydrodiol. The structures and sterochemistry of the dihydrodiols were established by comparisons of their UV spectra and chromatographic characteristics using HPLC with those of authentic compounds or, when no authentic compounds were available, by UV, NMR and mass spectral analysis. An examination by HPLC of the dihydrodiols formed in the metabolism, by rat-liver microsomal fractions, of benz[a] anthracene and 7,12-dimethylbenz[a] anthracene was carried out. The metabolic dihydriols were identified by comparisons of their chromatographic and UV or fluorescence spectral characteristics with compounds of known structures. The principle metabolic dihydriols formed from both benz[a] anthracene and 7,12-dimethylbenz[a] anthracene were the trans-5,6- and trans-8,9-dihydrodiols. The 1,2- and 10,11-dihydrodiols were identified as minor products of the metabolism of benz [a] anthracene and the tentative identification of the trans-3,4-dihydriol as a metabolite was made from fluorescence and chromatographic data. The minor metabolic dihydriols formed from 7,12-dimethylbenz[a] anthracene were the trans-3,4-dihydrodiol and the trans-10,11-dihydriol but the trans-1,2-dihydrodiol was not detected in the present study.     

Regio- and Stereoselective Metabolism of 7,12-Dimethylbenz[a]anthracene by Mycobacterium vanbaalenii PYR-1

The degradation of 7,12-dimethylbenz[a]anthracene (DMBA), a carcinogenic polycyclic aromatic hydrocarbon, by cultures of Mycobacterium vanbaalenii PYR-1 was studied. When M. vanbaalenii PYR-1 was grown in the presence of DMBA for 136 h, high-pressure liquid chromatography (HPLC) analysis showed the presence of four ethyl acetate-extractable compounds and unutilized substrate. Characterization of the metabolites by mass and nuclear magnetic resonance spectrometry indicated initial attack at the C-5 and C-6 positions and on the methyl group attached to C-7 of DMBA. The metabolites were identified as cis-5,6-dihydro-5,6-dihydroxy-7,12-dimethylbenz[a]anthracene (DMBA cis-5,6-dihydrodiol), trans-5,6-dihydro-5,6-dihydroxy-7,12-dimethylbenz[a]anthracene (DMBA trans-5,6-dihydrodiol), and 7-hydroxymethyl-12-methylbenz[a]anthracene, suggesting dioxygenation and monooxygenation reactions. Chiral stationary-phase HPLC analysis of the dihydrodiols showed that DMBA cis-5,6-dihydrodiol had 95% 5S,6R and 5% 5R,6S absolute stereochemistry. On the other hand, the DMBA trans-5,6-dihydrodiol was a 100% 5S,6S enantiomer. A minor photooxidation product, 7,12-epidioxy-7,12-dimethylbenz[a]anthracene, was also formed. The results demonstrate that M. vanbaalenii PYR-1 is highly regio- and stereoselective in the degradation of DMBA.     

Additional evidence for the involvement of the 3,4-diol 1,2-oxides in the metabolic activation of 7,12-dimethylbenz[a]anthracene in mouse skin

The role of vicinal diol-epoxides in the metabolic activation of 7,12-dimethylbenz[a]anthracene to intermediates that react with nucleic acids was investigated using Sephadex LH-20 column chromatography and high pressure liquid chromatography. The results show that some of the hydrocarbon-DNA products formed in mouse skin treated in vivo with 7,12-dimethylbenz[a]anthracene arise from the reaction of DNA with 3,4-dihydro-3,4-dihydroxy-7,12-dimethylbenz[a]anthracene 1,2-oxides which, on the basis of this and other evidence, appears to be a biologically-active metabolite of 7,12-dimethylbenz[a]anthracene. However, since other nucleic acid-hydrocarbon adducts were also present that have not been identified as resulting from the reaction of the 3,4-diol 1,2-oxides with DNA, other mechanisms may also be involved in the metabolic activation of 7,12-dimethylbenz[a]anthracene in mouse skin.     

The mechanism of microsomal hydroxylation of 7-methylbenz(a)-anthracene and 7,12-dimethylbenz(a)anthracene by oxygen-18 studies

The carcinogenic 7-methylbenz[a]anthracene and 7,12-dimethylbenz[a]anthracene were converted by rat liver microsomes into the corresponding hydroxymethyl derivatives and other metabolic products. The 7-methylbenz[a]anthracene incubation was carried out in H₂¹⁸O, and no incorporation of oxygen-18 was found in the hydroxymethyl metabolite isolated and purified by high pressure liquid chromatography, and analyzed by mass spectrometry. When 7-methylbenz[a]anthracene or 7,12-dimethylbenz[a]anthracene was incubated with ¹⁸O₂, isotope incorporation was observed in the corresponding hydroxymethyl derivatives, indicating that such hydroxylation is a true oxygenase reaction.     

Effect of cobaltous chloride on spontaneous mutation induction in a Bacillus subtilis mutator strain

The antimutagenic effects of selenium as sodium selenite were investigated using the Ames Salmonella/microsome mutagenicity test. The compounds examined were acridine orange and 7,12-dimethylbenz[a]anthracene. Selenium (22 ppm) reduced the number of histidine revertants caused by 20 μg acridine orange and 20 μg 7,12-dimethylbenz[a]anthracene by 52 and 74%, respectively. Increasing the quantity of selenium added to the plates further suppressed the mutagenicity of the test compounds. The antimutagenic effects of selenium cannot be explained by lethality of Salmonella typhimurium.     

Characterization of a photoproduct of 7,12-dimethylbenz[alpha]anthracene and its effects on chick-embryo cells in culture.

A common impurity of 7,12-dimethylbenz[alpha]anthracene was more effective than 7,12-dimethylbenz[alpha]anthracene in inducing morphological alterations, and in causing an increase in glucose uptake, DNA synthesis and cell number in chick-embryo fibroblasts. Gradual morphological transformation follows the increase in DNA synthesis after 2 days when either primary or secondary cultures are treated with 3 microgram of the compound/ml. The compound, isolated from 7,12-dimethylbenz[alpha]anthracene by alumina column chromatography, was characterized by t.l.c., mass spectroscopy, carbon-hydrogen analysis, u.v. and nuclear-magnetic-resonance spectroscopy and thermal decomposition. It was the photo-oxidation product of 7,12-dimethylbenz[alpha]anthracene, 7,12-epidioxy-7,12-dimethylbenz[alpha]anthracene. It is suggested that some of the biological effects observed after treatment of cultures with 7,12-dimethylbenz[alpha]anthracene may be due in part to the presence of the photo-oxidation product.     

Enhancement of aryl hydrocarbon hydroxylase activity in cultured rodent cells by X-irradiation.

X-irradiation (500 rads) was found to enhance the aryl hydrocarbon hydroxylase (AHH) activity of three cell lines. Radiation followed by induction with benz (a) anthracene (5–15 μg/ml) produced a synergistic effect on AHH. These effects were highly significant and were observed most dramatically with a hamster tumor cell line, A(T_l)Cl-3,a nd to a lesser extent in secondary hamsters embryo cells and mouse $\text{C}\text{3}\text{H}\text{/10}\text{T}\text{1}\text{2}$ CL8 cells.     

Orientation of methanolic attack on the K-region epoxide of 7,12-dimethylbenz (a) anthracene

Methanolysis of the K-region epoxide of 7,12-dimethylbenz (a) anthracene (DMBA) gave rise to two hydroxy-methoxy derivatives which were dehydrated to 5-methoxy-DMBA and 6-methoxy-DMBA at a ratio of 5:1. The data indicate that methanol attacks preferentially at the 5-position of the arene oxide. These results are in accord with steric considerations of reactivity.     

The effect of the bay-region 12-methyl group on the stereoselective metabolism at the K-region of 7,12-dimethylbenz[a]anthracene by rat liver microsomes.

The enantiomers of a trans-5,6-dihydrodiol formed in the metabolism of 7,12-dimethylbenz[a]anthracene by rat liver microsomes (microsomal fractions) were resolved by chiral stationary-phase high-performance liquid chromatography. The major 7,12-dimethylbenz[a]anthracene trans-5,6-dihydrodiol enantiomer and its hydrogenation product 5,6,8,9,10,11-hexahydro-trans-5,6-diol were found to have 5S,6S absolute configurations by the exciton chirality c.d. method. The R,R/S,S enantiomer ratios of 7,12-dimethylbenz[a]anthracene trans-5,6-dihydrodiol formed in the metabolism of 7,12-dimethylbenz[a]anthracene by liver microsomes from untreated, 3-methylcholanthrene-treated and phenobarbital-treated male Sprague-Dawley rats were found to be 11:89, 6:94, and 5:95 respectively. These findings and those reported previously on the metabolic formations of trans-5,6-dihydrodiols from 7-methylbenz[a]anthracene and 12-methylbenz[a]anthracene suggest that the 12-methyl group in 7,12-dimethylbenz[a]anthracene plays an important role in determining the stereoselective metabolism at the K-region 5,6-double bond. Furthermore, the finding that formation of 5S,6S-dihydrodiol as the predominant enantiomer was not significantly affected by the isoenzymic composition of cytochrome P-450 present in microsomes prepared from the livers of the rats pretreated with the different inducing agents indicates that the stereoselectivity depends on the substrate metabolized rather than on the precise nature of the metabolizing-enzyme system.     

The involvement of a non-'bay-region' diol-epoxide in the formation of benz(a)anthracene-DNA adducts in a rat-liver microsomal system

The metabolic activation of benz(a)anthracene was investigated by incubating [³H]-benz(a)anthracene with DNA, a NADPH-generating system and rat-liver microsomes. When hydrolysates of the DNA were chromatographed on Sephadex LH20 columns, three hydrocarbon-nucleoside adduct peaks were resolved and these were further examined using HPLC. One adduct probably results from the reaction of the non-bay-region diol-epoxide $\text{r}$-8,$\text{t}$-9-dihydroxy-$\text{t}$-10,11-oxy-8,9,10,11-tetrahydrobenz(a)anthracene $\text{(}\text{anti}$-BA-8,9-diol 10,11-oxide) with DNA. The other two adducts did not co-chromatograph with adducts formed from any of the four possible isomeric diolepoxides that can be formed in the 8,9,10,11-ring of benz(a)anthracene.