Tumorigenicity of enantiomers of chrysene 1,2-dihydrodiol and of the diastereomeric bay-region chrysene 1,2-diol-3,4-epoxides on mouse skin and in newborn mice.

The tumorigenicity of chrysene, (+)and (-)-frans-1,2-dihydroxy-1,2-dihydrochrysene (chrysene 1,2-dihydrodiol), and the ( + )and (—)-enantiomers of the diastereomeric bay-region chrysene 1,2-diol-3,4-epoxides was assessed in two tumor models. A single topical application of 0.4 or 1.2 /¿mol of the chrysene derivatives on mouse skin followed by 25 weeks of promotion with 12-O-tetradecanoylphorbol-13-acetate re vealed that ( — )-chrysene 1,2-dihydrodiol had approximately twice the tumor-initiating activity of chrysene, while the (+)enantiomer had no significant tumorigenic activity. Of the four isomers of chrysene 1,2-diol-3,4-epoxide, only ( + )-1/?,2a-dihydroxy-3a,4a-epoxy-1,2,3,4-tetrahydrochrysene [( + )-diolepoxide 2], in which the benzylic hydroxyl group and epoxide oxygen are frans, had significant tumorigenic activity. The (+)-diol-epoxide 2 isomer had tumor-initiating activity that was equivalent to the activity of the parent hydrocarbon. In newborn mice, a total dose of 0.7 or 1.4 jumol of compound divided into three i.p. injections was administered on the first, eighth, and 15 days of life, and tumorigenic activity was determined when the mice were 37 to 41 weeks old. Control animals developed an average of 0.13 lung tumor/mouse, whereas animals treated with (+)-diol-epoxide 2, (-)-chrysene 1,2-dihydrodiol, or racemic chrysene 1,2-dihydrodiol had an average of 7.8, 6.5, and 2.5 lung tumors per mouse per jumol of compound administered, respectively. The parent hydrocarbon and all other derivatives tested were essentially inactive in producing lung tumors. With respect to hepatic tumors in male mice, (—)-chrysene 1,2-dihydrodiol was the most active compound tested, and produced 3-, 4-, 6-, and 10-fold more hepatic tumors per mouse per /imol of compound administered than did ( + )-diol-epoxide 2, racemic chrysene 1,2-dihydrodiol, chrysene, and ( + )-chrysene 1,2-dihydrodiol, respectively. Other derivatives had no significant hepatotumorigenic activity at the doses tested. The high tumorigenic activity of ( —)-chrysene 1,2-dihydrodiol and ( + )-diol-epoxide 2 on mouse skin and in newborn mice indicate that these compounds are, respectively, proximate and ultimate carcinogenic metabolites of chrysene. The (+)-diol-epoxide 2 isomer of chrysene has absolute configuration such that it is superimposable on the (+)-diol-epoxide 2 isomer of benzo(a)pyrene [(+)-7/î,8a-dihydroxy-9a,10a-epoxy-7,8,9,10-tetrahydrobenzo(a)pyrene], the ultimate carcinogenic metabolite of benzo(a)pyrene. INTRODUCTION Bay-region diol-epoxides are the only known class of ultimate carcinogens formed from polycyclic aromatic hydrocarbons (3, 10). These diol-epoxides are derived metabolically from benzoring frans-dihydrodiols with a bay-region double bond and can be formed as a pair of diastereomers (14) in which the benzylic hydroxyl group is either cis (diol-epoxide 1)2 or frans (diolepoxide 2) to the epoxide oxygen.3 With one exception (6), the bay-region diol-epoxide 2 isomers have much greater tumori genic activity on mouse skin and in newborn mice than do their diol-epoxide 1 counterparts (3, 10). Since each diastereomeric diol-epoxide can be resolved into a pair of enantiomers, a total of 4 stereoisomers of a bay-region diol-epoxide is metabolically possible (cf. Chart 1). Only in the case of benzo(a)pyrene has the tumorigenic activity of the optically pure isomers been evaluated (2,12). These studies established that essentially all of the tumorigenic activity resides in the (+)-diol-epoxide 2 isomer. It is noteworthy that this is also the predominant isomer formed when benzo(a)pyrene is metabolized by the cytochrome P-450-dependent monooxygenase system and epoxide hydrolase (5, 15, 23). In order to further evaluate the significance of stereochemical factors in the expression of tumorigenic activity by polycyclic aromatic hydrocarbons, the individual isomers of other bayregion diol-epoxides must also be tested in a similar fashion. The present report evaluates the influence of stereochemical factors on the tumorigenicity of chrysene. The data presented here, together with previous metabolic studies (9,16), establish that only the metabolically predominant ( + )-diol-epoxide 2 isomer of chrysene possesses high tumorigenic activity on mouse skin and in newborn mice. MATERIALS AND METHODS Chemicals. Chrysene was purchased from Aldrich Chemical Co., Milwaukee, Wis., and was purified by several recrystallizations from 1To whom requests for reprints should be addressed. Received May 19, 1982; accepted August 31. 1982. 2 The abbreviations used are; (-)-chrysene 1,2-diol-3,4-epoxide 1, (-M1R, 2S, 3R 4SH/3,2a-dihydroxy-3;8,4/3-epoxy-1.2,3,4-tetrahydrochrysene; (+)chrysene 1,2-diol-3.4-epoxide 1. (+M1S, 2fl, 3S, 4R)-1a,2/î-dihydroxy-3a,4aepoxy-1,2,3,4-tetrahydrochrysene; (-)-chrysene 1,2-diol-3,4-epoxide 2, (-)<1S, 2W, 3fl, 4SMa,2/?-dihydroxy-3/9,4/J-epoxy-1,2,3,4-tetrahydrochrysene; (+)-chrysene 1,2-diol-3,4-epoxide 2, (+M1R 2S, 3S, 4R)-1/3,2a-dihydroxy3a.4a-epoxy-1,2,3,4-tetrahydrochrysene; ( + )-chrysene 1,2-dihydrodiol, (+)(1S,2S)-1o,2/3-dihydroxy-1,2-dihydrochrysene; (-)-chrysene 1,2-dihydrodiol, (-)-(1fl,2W)-1/6,2a-dihydroxy-1,2-dihydrochrysene; DMSO, dimethyl sulfoxide. In the isomer 1 series of the diol-epoxides, the benzylic hydroxyl group and the epoxide oxygen are cis, whereas in the isomer 2 series, these 2 groups are frans. 3 The relative stereochemistry of the hydroxyl groups of dihydrodiols formed by microsomal epoxide hydrolase is trans. The present study deals exclusively with isomers of frans-dihydrodiols and bay-region diol-epoxides derived from these dihydrodiols. 192 CANCER RESEARCH VOL. 43 Research. on January 12, 2018. © 1983 American Association for Cancer cancerres.aacrjournals.org Downloaded from Tumorigenicity of Chrysene Derivatives (-)-(IR,2S,3R.4S)-l/3,2a-DIOL3ß,4/3-EPOXIDE-l (•H-(IS,2R,3S,4R)-l*,2/3-DIOL3a,4d-EPOXIDE-l (-)-(IS,2R,3R,4S)-la,2/5-DIOL3j9,4/3-EPOXIDE-2 (-H-(IR,2S,3S,4Rl-l#,2a-DIOL3o,4a-EPOXIDE-2 (+>-(IS,2S)-la,2/3-DIHYDRODIOL (-)-( IR,2R)-l/3,2a-DIHYDRODIOL Chart 1. Absolute configuration of the enantiomers of chrysene 1,2-dihydrodiol and the optically active diastereomers of the bay-region 1,2-diol-3.4-epoxides. benzene prior to use (m.p. 256°). Racemic chrysene 1,2-dihydrodiol was obtained by unequivocal chemical synthesis as described (4). Optically pure ( + )and (-)-enantiomers of the diastereomeric chry sene 1,2-diol-3,4-epoxides were synthesized from the (+)and (-)enantiomers of chrysene 1,2-dihydrodiol, which were obtained via resolution of frans-1,2-dihydroxy-1,2,3,4-tetrahydrochrysene as de scribed (22). DMSO was distilled from calcium hydride under reduced pressure and stored under an argon atmosphere in amber bottles. 12O-Tetradecanoylphorbol-13-acetate was purchased from Chemical Carcinogenesis, Inc., Eden Prairie, Minn. The chrysene 1,2-diol-3,4epoxides were dissolved in DMSO and are stored at -90°. Repeated thawing and freezing of the samples did not result in any decrease in their mutagenic activity in Salmonella typhimurium, indicating their stability in organic solvent. Tumorigenicity Studies on Mouse Skin. Female Charles River CD1 mice were obtained at 7 to 8 weeks of age from Charles River Mouse Farms, North Wilmington, Mass. The mice were shaved on the dorsal surface with electric clippers. Two days later, 30 mice in each treatment group were given a single topical application of the chrysene deriva tives in 200 ;ul of solvent (10% DMSO in acetone). Control animals received only solvent. The tumor promoter 12-O-tetradecanoylphorbol13-acetate (16 nmol/200 jul of acetone) was applied twice weekly to each mouse, beginning 7 days after application of initiator or solvent. Development of skin tumors was recorded once every 2 weeks, and papillomas greater than 2 mm in diameter were included in the cumu lative total when they persisted for 2 weeks or longer. Statistical significance of skin tumor data was determined by the method of Mainland and Murray (8) and by Student's f test. Newborn Mouse Experiments. Pregnant mice of the Swiss-Webster BLU:Ha (ICR) strain (Blue Spruce Farms, Altamont, N. Y.) were housed in plastic cages on corncob bedding. They delivered their litters from 2 to 6 days after arrival. Within 24 hr of birth, 10 pups in each litter were given i.p. injections of the first dose of compound. The mice were given a total dose of 0.7 or 1.4 mmol of compound divided into 3 injections of 0.1, 0.2, and 0.4 pmol, or 0.2, 0.4, and 0.8 jimol, respectively. The compounds were administered on the first, eighth, and 15th day of life, respectively. Control mice were given 3 injections of DMSO (5, 10, and 20 jil). The mice were weaned at 23 days of age and killed at 37 to 41 weeks of age. At necropsy, the major organs of each animal were examined grossly, tumors were counted, and tissues were fixed in 10% buffered formalin. A representative number of pulmonary tumors and all hepatic tumors were examined histologically. Pathology of the lung (11 ) and liver (17, 18) tumors was the same as has been described previously. Statistical significance of the newborn mouse tumor data was evaluated by the Fisher 2x2 exact test and the Mann-Whitney U test.