Tumorigenic Activity of 3-Methyicholanthrene Metabolites on Mouse Skin and in Newborn Mice

INTRODUCTION 3-Methylcholanthrene and eight of its metabolites were tested for tumonigenic activity in two tumor models. In the tumonigenicity study on mouse skin, a single topical application of 3 to 30 nmol of compound was followed 7 days later by twice weekly applications of the tumor promotor i 2-0-tetra decanoylphorbol-i 3-acetate for 30 weeks. Comparisons of the percentage of mice with tumors and the average number of tumors per mouse indicated that 3-methylcholanthrene, 2-hy droxy-3methylcholanthrene, 3-methylcholanthrene2-one, and a i ,9,i 0-tnihydroxy-9,i 0-dihydro-3-methylcholanthrene diastereomer were approximately equipotent as tumor initia tons. i -Hydroxy-3-methylcholanthrene had approximately one fourth the tumor-initiating activity of the most active com pounds, and 3-methylcholanthrene-i -one and trans-i i ,i 2-di hydroxy-i 1,i 2-dihydro-3-methylcholanthrene had no signifi cant tumonigenic activity at the doses tested. In the tumori genicity study in newborn mice, increasing amounts of the compounds were injected i.p. on the i st, 8th, and i 5th days of life to give a total dose of 2i or 49 nmol. The experiment was terminated when the animals were 35 to 39 weeks old. With respect to pulmonary tumors, i ,9,i 0-trihydroxy-9,i 0-dihydro 3-methylcholanthrene was the most tumonigenic compound tested at both doses. 3-Methylcholanthrene and 3-methylchol anthrene-2-one were the next most active compounds, and they had approximately one-half to one-third of the activity of i ,9, 10-tnihydnoxy-9,i 0-dihydro-3-methylcholanthrene when the data were expressed as pulmonary tumors per mouse. 2Hydroxy-3-methylcholanthrene was slightly less active than were 3-methylcholanthrene and 3-methylcholanthrene-2-one. i -Hydroxy-3-methylcholanthnene had marginal tumorigenic ad tivity. 3-Methylcholanthrene i i ,i 2-oxide, trans-i i ,i 2-dihy droxy-i 1,i 2-dihydro-3-methylcholanthrene, and 3-methyl cholanthrene-i -one were inactive at the doses tested. i ,9, i 0-Tnihydroxy-9, i 0-dihydro-3-methylcholanthrene at the 2iand 49-nmol doses also produced hepatic tumors in 50 and 8i % of the male mice, respectively. 3-Methylcholan threne and 2-hydroxy-3-methylcholanthrene had one-fourth to one-tenth the activity of i ,9,i 0-tnihydroxy-9,i 0-dihydro-3methylcholanthrene in the liver. The other 3-methylcholan threne metabolites were essentially inactive in producing he patic tumors. The high tumorigenic activity of i ,9,i 0-trihy droxy-9,i 0-dihydno-3-methylcholanthrene on mouse skin and in newborn mice provides evidence for bay-region activation of 3-methylcholanthrene to an ultimate carcinogen. The data also indicate that other metabolites of 3-methylcholanthrene may play a role in the carcinogenicity of this polycyclic aromatic hydrocarbon. @ To whom requests for reprints should be addressed. Received January 25. 1979: accepted June 8, 1979. Previous studies from our laboratories have been concerned with the identification of ultimate carcinogenic metabolites of the unsubstituted polycyclic aromatic hydrocarbons benzo(a)pyrene, benzo(a)anthracene, chrysene, and dibenzo (a,h)anthnacene (5, i 2, 16). These studies have shown that dihydrodiols on angular benzo rings with †̃ †̃bay-region' †̃ double bonds are proximate carcinogens and that diol-epoxides formed from these dihydnodiols, in which the epoxide forms part of the bay region of the hydrocarbon, are ultimate carci nogenic metabolites. Among the alternant polycyclic aromatic hydrocarbons, several alkyl-substituted derivatives of the weak carcinogen benzo(a)anthnacene, such as MC,2 7-methyl benzo(a)anthracene, 7,12-dimethylbenzo(a)anthracene, and F-nor-steranthrene, are potent carcinogens (1 , 7, 15). Recent studies have suggested that bay-region diol-epoxides of certain alkyl-substituted polycyclic aromatic hydrocarbons are also prime candidates as ultimate carcinogenic metabolites (4, 9, i 3, i 4, i 7—i 9, 25, 27, 29, 32). Such alkyl substituents not only are subject to metabolism themselves but also can dimin ish the rate at which monooxygenase enzymes form arene oxides at the formal aromatic double bonds to which they are attached. The resultant increases or decreases in the formation of positional isomers of dihydrodiols can thereby modulate the carcinogenic potency of the parent hydrocarbon (i 0, 1i). To date, several oxygenated metabolites of the potent car cinogen MC have been identified (22—26),but the structure(s) of the ultimate carcinogenic metabolite(s) has yet to be deter mined. Since MC is an alkyl-substituted derivative of benzo(a)anthracene, we sought to determine if this polycyclic aromatic hydrocarbon was metabolically activated to an ulti mate carcinogenic metabolite in a manner analogous to benzo(a)anthnacene, i.e. , a bay-region diol-epoxide, which in the case of MC would be a 9, i 0-diol-7,8-epoxide. Although our in vitro metabolism studies with rat liver microsomes mdi dated that dihydrodiol formation was a minor metabolic path way for MC (24, 25), a major primary oxidative metabolite of the hydrocarbon, i -hydroxy-MC, was metabolically converted to at least 4 dihydnodiols to the extent of i 0 to 20% of total metabolism (25). The 2 major dihydrodiols formed from racemic i -hydroxy-MC by rat liver microsomes have been identified as diastereomerically related trans-9, 10-dihydrodiols.3 These 2 The abbreviations used are: MC, 3-methylcholanthrene: 1 -hydroxy-MC, 1 hydroxy-3-methylcholanthrene; 1-hydroxy-MC 9, 10-dihydrodiol, 1.9,10-tnihy droxy-9, 10-dihydro-3-methylcholanthrene in which the 9and 10-hydroxyl groups have the trans configuration: 2-hydroxy-MC, 2-hydroxy-3-methylcholan threne; 1-keto-MC, 3-methylcholanthrene-1 -one; 2-keto-MC, 3-methylcholan threne-2-one; MC 11.12-oxide, 3-methylcholanthrene 11.12-oxide; MC-1 1,12dihydrodiol, trans-i 1.12-dihydroxy-1 1.12-dihydro-3-methylcholanthrene; TPA, 12-O-tetradecanoylphorbol-1 3-acetate. Unless stated otherwise, all compounds are racemic mixtures where optical enantiomers are possible. @ Since racemic 1-hydroxy-MC was used as a substrate for metabolism studies