Brian Tierney

The metabolic activation of 7-methylbenz(a)anthracene in mouse skin

The metabolism of 7-methylbenz(a)anthracene by rat-liver preparations and by mouse skin has been studied using a combination of thin-layer and high pressure liquid chromatography and all five possible trans-dihydrodiols have been detected as metabolites but in different proportions. The roles of these dihydrodiols and of the related vicinal diol-epoxides in the metabolic activation of 7-methylbenz(a)anthracene in mouse skin has been studied using Sephadex LH-20 column chromatography. The results show that the hydrocarbon-nucleic acid products formed in mouse skin in vivo most probably arise from 3,4-dihydro-3,4-dihydroxy-7-methylbenz(a)anthracene 1,2-oxide which, on the basis of this and other evidence, appears to be the reactive intermediate involved in the metabolic activation of 7-methylbenz(a)anthracene in this tissue.

The identity and nuclear uptake of a cytosolic binding protein for 3-methylcholanthrene

Abstract The ability of 3-methylcholanthrene to interact noncovalently with rat liver cytosolic proteins was studied using Sephadex G200 chromatography. A specific 3-methylcholanthrene binding fraction from Sephadex G200 chromatography, termed peak B, when incubated with rat liver nuclei was able to translocate 3-methylcholanthrene into the nucleus. This translocation occurred faster and was quantitatively greater than the binding of 3-methylcholanthrene in buffer to nuclei. In addition, the nuclear uptake of peak B was increased by prewarming, suggesting that a heat-sensitive activation step may occur prior to the translocation process. However, no evidence was found on sucrose gradients for any conformational change in the protein fraction studied here. The translocation to the nucleus was temperature and time dependent. An examination of the characteristics of this 3-methylcholanthrene binding protein using Sephacryl S200 column chromatography showed a small number of high-affinity, saturable, binding sites to be present. These had an apparent dissociation constant, Kd, of 2.8 n m and a binding capacity of 770 fmol/mg of cytosolic protein. The selectivity of this protein was examined by competition studies and, in general, polycyclic hydrocarbons competed for the binding site, except for anthracene and phenanthrene. Of the inducers studied, 5,6-benzoflavone was a strong competitor. No competition was found with 12-O-tetradecanoyl phorbol-13-acetate, 2,6-ditertbutyl-p-cresol, β-retinyl acetate, or a number of steroids, except for 17β-estradiol which exhibited moderate binding. Peak B had a sedimentation coefficient of 4.2 S when analyzed on a linear sucrose gradient. Chromatography of peak B on a calibrated Sephacryl S200 column gave a molecular weight corresponding to 44,600 ± 4000.

High microsome-mediated mutagenicity of the 3,4-dihydrodiol of 7-methylbenz[a]anthracene in S. typhimurium TA 98

Abstract 7-Methylbenz[a]anthracene and the 1,2-, 3,4-, 5,6- and 8,9-dihydrodiols derived from this hydrocarbon have been tested for mutagenicity towards S. typhimurium TA 98 in the presence of rat-liver post-mitochondrial supernatant. At non-toxic concentrations, the mutagenicity of the non-K-region 3,4-dihydrodiol was more than ten-fold higher than that of the other K-region and non-K-region dihydrodiols and more than three-fold higher than that of the parent hydrocarbon. 1,1,1-Trichloropropene 2,3-oxide, an inhibitor of epoxide hydratase, increased the microsome-mediated mutagenicity of 7-methylbenz[a]anthracene but did not alter that of the four related dihydrodiols.

The metabolic activation of 7-methylbenz(a)anthracene in mouse skin: High tumour-initiating activity of the 3,4-dihydrodiol

Summary 7-Methylbenz(a)anthracene and four related dihydrodiol metabolites were tested for their abilities to initiate tumour formation on mouse skin. A single application (25 μg) of the hydrocarbon or of the related 1,2-, 3,4-, 5,6- and 8,9-diols was made to the dorsal skin of CDI mice and papilloma formation promoted by thrice-weekly applications of 12-O-tetradecanoyl-phorbol-13-acetate. The 3,4-dihydrodiol was the most active compound tested, 7-methyl-benz(a)anthracene and the 8,9-diol were less active and the 1,2- and 5,6-diols were almost inactive. These results support the conclusion that, with 7-methyl-benz(a)anthracene, the active diol-epoxide is 3,4-dihydro-3,4-dihydroxy-7-methylbenz(a)anthracene 1,2-oxide.

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.

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

Abstract 7,12-Dimethylbenz[a]anthracene and its 3,4-, 5,6-, 8,9- and 10,11-dihydrodiols have been tested for mutagenicity towards S . 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.

Induction of sister-chromatid exchanges in Chinese hamster ovary cells treated in vitro with non-k-region dihydrodiols of 7-methylbenz[a]anthracene and benzo[a]pyrene

Studies were carried out on the incidence of sister-chromatid exchanges induced in Chinese hamster ovary cells by in vitro treatment with the polycyclic aromatic hydrocarbons 7-methylbenz[a]anthracene and benzo[a]pyrene and with related K-region and non-K-region dihydrodiols. Appreciable increased in the incidence of sister-chromatid exchanges were apparent in cells treated with non-K-region dihydrodiols: the most active compounds were 3,4-dihydro-3,4-dihydroxy-7-methylbenz[a]anthracene and 7,8-dihydro-7,8-dihydroxybenzo[a]pyrene and the effects were dose-dependent. The parent hydrocarbons and the related K-region dihydrodiols induced some sister-chromatid exchanges but they were considerably less active than these two non-K-region diols. The results suggest that this system may usefully be applied to studies aimed at determining which dihydrodiols are important in the metabolic activation of the carcinogenic polycyclic hydrocarbons. These and other results also infer that Chinese hamster ovary cells possess some intrinsic ability to metabolize such compounds in the absence of exogenous activation systems.

The preparation of dihydrodiols from 7-methylbenz[a]anthracene

The products formed when the carcinogenic polycyclic hydrocarbon 7-methylbenz[a] anthracene is oxidized with an ascorbic acid-ferrous sulphate mixture have been investigated. All 5 possible dihydrodiols were formed and the isolation of the 3 non-K-region dihydrodiols, trans-1,2-dihydro-1,2-dihydroxy-7-methylbenz[a]anthracene, trans-3,4-dihydro-3,4-dihydroxy-7-methylbenz[a] anthracene and trans-8,9-dihydro-8,9-dihydroxy-7-methylbenz[a] anthracene is described. The purification of the dihydrodiols was carried out by thin-layer (TLC) followed by preparative high pressure liquid chromatography (HPLC). The ultra-violet, spectral and nuclear magnetic resonance (NMR) characteristics of the dihydrodiols are reported and the data used to assign the proposed structures. An explanation for the unusual preferred conformation which the 8,9-dihydrodiol adopts is advanced.

The formation of dihydrodiols by chemical or enzymic oxidation of 3-methylcholanthrene

The chemical oxidation of 3-methylcholanthrene in an ascorbic acid-ferrous sulphate-EDTA reaction mixture gave all five possible dihydrodiols. The structures and stereochemistry of the dihydrodiols were shown by UV, mass and NMR spectral studies and by chemical examination to be cis-2a,3-dihydroxy-3-methylcholanthrene, trans-4,5-dihydro-4,5-dihydroxy-3-methylcholanthrene, trans-7,8-dihydro-7,8-dihydroxy-3-methylcholanthrene, trans-9,10-dihydro-9,10-dihydroxy-3-methylcholanthrene, cis-11,12-dihydro-11,12-dihydroxy-3-methylcholanthrene and trans-11,12-dihydro-11,12-dihydroxy-3-methylcholanthrene. An examination by HPLC of the dihydrodiols formed in the metabolism of 3-methylcholanthrene by rat-liver microsomal preparations showed the presence of trans-4,5-dihydro-4,5-dihydoxy-3-methylcholanthrene, trans-7,8-dihydro-7,8-dihydroxy-3-methylcholanthrene, trans-9,10-dihydro-9,10-dihydroxy-3-methylcholanthrene and trans-11,12-dihydro-11,12-dihydroxy-3-methylcholanthrene, identified by comparison of their UV and chromatographic characteristics with those of authentic standards. Tentative identification of cis- and trans-1,2-dihydroxy-3-methylcholanthrene, cis-2a,3-dihydroxy-3-methylcholanthrene and cis-11,12-dihydro-11,12-dihydroxy-3-methylcholanthrene as metabolites were made from their mobilities using HPLC. A quantitative comparison of the dihydrodiols formed from 3H-labelled 3-methylcholanthrene by microsomal preparations from the livers of normal and 3-methylcholanthrene-treated rats was carried out. trans-9,10-Dihydro-9,10-dihydroxy-3-methylcholanthrene and cis- and trans-1,2-dihydroxy-3-methylcholanthrene were formed when 3-methylcholanthrene was incubated with mouse skin in organ culture.

Differences in the binding of 3-methylcholanthrene and phenobarbital to rat liver cytosolic and nuclear protein fractions

Abstract The inducers of cytochrome P -450c and P -450b, 3-methylcholanthrene and phenobarbital, respectively, have been studied in their interaction with subcellular fractions from rat liver. 3-Methylcholanthrene bound to both nuclear and cytoplasmic components as demonstrated by DNA-cellulose chromatography. The binding of 3-methylcholanthrene to cytosolic proteins, on DNA-cellulose, was approximately 27 fmol/mg of applied protein, whereas the binding to nuclear proteins was 250–570 fmol/mg applied protein. Phenobarbital did not bind to proteins of rat serum, rat liver cytosol, or rat liver nuclei which could bind to DNA-cellulose. Further examination of the potential interaction of phenobarbital to rat liver cytosolic proteins was carried out using either DEAE A-50 Sephadex chromatography, charcoal dextran analysis, or sucrose density gradients. No binding of phenobarbital to rat liver cytosolic proteins was observed under these experimental conditions. In contrast, the binding of 3-methylcholanthrene to cytosolic proteins showed four peaks of radioactivity after DEAE A-50 Sephadex chromatography, two peaks by sucrose density gradient analysis, and specific binding (0.13 pmol/mg protein) was observed using the charcoal dextran technique. One of the peaks on sucrose gradients was labile in the presence of salt. The uptake and intranuclear distribution of 3-methylcholanthrene and phenobarbital were markedly different after incubation with whole nuclei: 64% of the available 3-methylcholanthrene but only 3% of the available phenobarbital radioactivity became associated with nuclei. Of this radioactivity, the highest specific activity of the 3-methylcholanthrene radioactivity was associated with the 2 m KCl-resistant nuclear pellet and the highest specific activity of the phenobarbital radioactivity was associated with the nuclear fraction soluble in the absence of salt. These results are interpreted in regard to the induction of cytochrome P -450c.