- Email: [email protected]
The importance of the “bay region” diol-epoxide in 7,12-dimethylbenz[a]anthracene skin tumor initiation and mutagenesis
Cancer Letters, 6 (1979) 213--220 © Ei~evierINortb-Ho]land S~i,entific Publishers Ltd.
213
THE I M P O R T A N C E O F THE " B A Y R E G I O N " D I O L - E P O X I D E I N 7,12D I M E T H Y L B E N Z | a ] 2 ~ N T H R A C E N E S K I N TUMO]~ I N I T I A T I O N A N D MUTAGENESIS*,** T.J. SLAGAt, E. HUBERMAN, J. DiGIOVANNIt t and G. GLEASON Biology Division, Oak Ridge National Laboratory, Oak Ridge, Tennessee 37830 (U.&A.) R.G. H A R V E Y Ben May Laboratory for Cancer Research, University o f Chicago, Chicago, Illinois 60637
(U.S.A.) (Received 5 October 1978) (Revised version received 22 November 195'8) (Accepted 5 December 1978)
SUMMARY T h e skin tumor-initiating and V 7 9 m u t a g e n i c activities of various deriw'~tives o f 7 , 1 2 - d i m e t h y l b e n z [ a ] a n t h r a c e n e (DMBA) were investigated to d e t e r m i n e w h a t possible cellular metabolite(s) m a y be responsible for its carcinogenivity a n d / o r mutagenict~y. 1-,2-,3.,4- and 5 - h y d r o x y D M B A were f o u n d t o be essentially inactive as skin t u m o r initiators whereas 9- and 1 0 - h y d r o x y D M B A h a d w e a k activity. The (±)-trans D M B A 8,9- and 5,6-dihydrodiols were also *Research sponsored jointly by the NIH under Interagcney Agreement Grant CA-20076 and by the U.S. Department of Energy, under contract W-7405-eng-26 with the Union Carbide Corporation. **After our manuscript was submitted for publication, Maleveille and coworkers reported the high mutagenie activity of the 3,4-dihydrodiol of DMBA in S. typhimurium TA100 in the presence of a rat liver metabolizing system which agrees with the data presented here. See, Malaveflle, C. Bartseh, H., Tierney, B., Grovar, P.L. and Sims, P. (1978). Miarosome~ mediated mutagenicities of the dihydrodio]s of 7,12.dirnethyihenz[a]anthraeene: High mutagenic activity of the 3,4-dihydrodiol. Bioehem. Biophys. Res. Commun., 83, 1468--1473. t To whom requests for reprints and correspondence shound be addrea~ed to: Biology Division, Oak Ridge National Laboratory, Post Office Box Y, Oak Ridge, Tennessee 37830, U.S.A. T1"Present add~.e~: McPadie Laboratory for Cancer Reseaxeh, University of Wisconsin Medical SchooL, Madison, Wisconsin, U.S.A. Abbreviations: BP, benzo[a]pyrene; DMBA, 7,12-dimethylbenz[a]anthracene; PAH, polycyclic aromatic hydrocarbons; 1-. 2-, 3-, 4-, 5-, 9- and 10-OH DMBA, various phenols of DMBA; DMBA 5,6-diol, (±)trans-5,6-dihydroxy~5,6-d~hydro.DMBA; DMBA 8,9-diol, (±)traas8,9-dihydroxy-8,9-dihydro-DMBA; DMBA 8,9-diol-.~.0,11-epoxlde, (±)trans-8~, 9~-dihydroxyI0~,11~-epoxy-8,9,10,11-tetrahydro-DMBA; 1-CH~-, 2-CH3-, 2-CH~, and 5-CH3-DMBA, either 1-, 2 or 5,7,12-trimethylbenz[a ]anthracene~ 1-, 2-, 5-, and 11-fl-DMBA, 1-, 2-, 5- and 11-flaoro-DMBA; TPA, 12-O-tetradecanoyi-phorbr~l- 13-acetate.
214
essentially inactiveas skin tumor initiatorsand (-+)-DMBA 8~,9~-diol-!0a,llaepoxide had weak skin tumor initiatingactivity.All of the above tested derivatives of D M B A were essentiallyinactive as mutagens in the cell-mediated or direct V 7 9 mutagenesis systems. A methyl or fluoro addition to the I, 2 or 5 positions almost completely blocked the skin tumor initiatingand V 7 9 mutagenic activitiesof DIVIBA, whereas a fluoro addition to position 11 did not. F r o m our da'~awe suggest that a 'bay region' diol-epoxide m a y be important in D M B A carcinogenicity and mutagenicity.
INTRODUCTION Recent evidence has revealed that 'bay region' diol-epoxides of benzo[a]pyrene (BP), benz[a]anthracene, chrysene, 7-methylbenz[a]anthracene and dibenz[a,h]anthracene are highly carcinogenic and mutagenie [1,2,6,8,10--12, 14--17,21,23--26,30--36]. Furthermore, in vitro and in vivo binding [3,13,18, 19,22,27,29] and metabolism [28,37] studies, as well as the above mu~agenesis and carcinogenesis studies, have led to the conclusion that a 'bay region' diolepoxide of BP is responsible for its c~_reinogenicity and mutagenicity. Since DMBA is one of the most potent carcinogenic PAH, it would be of interest to determine whether DMBA is also activated into a carcinogen and mut~gen via this pathway. Moschel et ~L. [20] have recently presented fluorescence data which implicates the 'bay region' diol.epoxide of DMBA in the interaction of DMBA with cellular DNA. In addition, binding of DMBA to cells in culture analyzed by LH20 chromatot,~aphy also suggests that a 'bay region' diol epoxide is involved in DMBA binding [3]. In this study we compared the skin tumor initiation in mice and mutagenesis in V79 cells of a 'non-bay region' diol epoxide, several diols, phenols, and methyl and fluoro derivatives of DMBA. The data suggest that a "bay region' diol epoxide may be important in DMBA carcinogenicity and mutagenicity. A 'bay region' occurs in a polycyclic hydrocarbon when an angularly ,~used benzo ring is present [!0,11]. MATERIALS AND METHODS Chemicals DMBA was purchased from Sigma Chemical Company, St. Louis, Missouri and was more than 99% pure. 12-O-tetradecanoyl-plhorbol.13-acetate (TPA) was obtained from Dr. P. Borcher~, Universil~y of Minnesota, Minneapolis, Minnesota. The DMBA diols and diol-epoxide were synthesized and purified as previously described [5]. The 1-, 2-, 5-, and ll-fI-DMBA, the 1-, 2-, and 5CH3DMBA and the 1-, 2-, 3-, 4-, 5-, 9-, and 10-OH DMBA were generous gifts from Professor M. Newman, Ohio State University, Columbus, Ohio. All o:[ the hydrocarbons were prepared in the dark or in yellow light immediately before use.
215 Tumor experiments Female CD1 mice were purchased from Charles River Farms, North Wilmington, Massachusetts. Mice, 7--9 weeks old, were shaved with surgical clippers 2 days before treatment and only those in the resting phase of the hair cycle were used. in the tumor experiments groups of 30 animals received a single topical application of the test compound, followed I week later by twice weekly applications of TPA. The incidence of papillomas was recorded weekly and they were removed at random for histological verific~.tion. The DMBA phenols, diols and fluoro and CH3 derivatives were applied j.opically at a dose of 200 nmol in acetone whereas the diol-epoxide of DMBA was applied topically at a dose of 200 nmol in tetrahydrofuran. Direct and cell-mediated mu tagenesis assay Ouabain resistance was tested by seeding 3 × 10 s V79 ceils in 4 ml of medium into a Petri dish without the hamster ceils for the direct assay [8], or on a 1-day-old monolayer of 5000 F~-irradiated normal golden hamster cells seeded at 2 × 106 cells/Petri dish for the cell-mediated assay [6,9]. The cells were cultured as previously described [9]. DMBA and derivatives were added 5 h after the V79 cells were seeded in 1 ml of medium. Incubated for 2 days and then dissociated with a trypsin-EDTA solution and seeded at 200 cells/Petri dish in 5 ml of medium to detet~fine cloning efficiency and at 10 s cells/Petri dish in 4 ml of medium to determine the frequency of ouabain-resistant mutants. For selection of the mutants, ouabain at a final concentration of 1 mM was added in 1 ml of medium 2 days after cell seeding, The colonies were counted after staining with Giemsa. Cloning efficiency was determined by counting the number of colonies in 6--8 Petri dishes/point at 7--8 days after cell seeding, and the frequency of ouabain-resistant mutants was determined by counting 24--32 Petri dishes]point at 14--16 days after cell seeding. The results were based on 2 experiments/point. The number of mutants after induction with DMBA or l l - f l - D M B A varied up to 30~d. In all other cases, the number of mutants in the different experiments varied up to 60%. RESULTS A N D DISCUSSION
Table 1 shows the relative skin tumor-initiating activities of the various derivatives of DMBA. The 1-, 2-, 3-, 4 and 5-OH DMBA were found not to have skin tumor-initiating activity whereas the 9- and 10-OH DMBA had weak activity. The 5,6- and 8,9-diols of DMBA were also found to be inactive and the (±)DMBA 88, 9~-diol-10a, ll~-epoxide had only weak skin tumor-initiating activity. It is interesting to point out that, as in the case of the tmnor experiments, DMBA and its ll-fluors derivatives were potent mutagens when tested in the cell mediated assay whii~e all other derivatives were either inactive or exhibited poor activity (Table I!). In the direct V79 mutagenesis assay in which there is no detectable polycyclk~ hydrocarbon metabolism neither DMBA nor any of the tested phenols, diols and diol-epoxide were active as mutagens at hydrocarbon concentration of up to 4 ~M.
216 TABLE 1 S K I N T U M O R INITIATING ACTIVITIES OF. V A R I O U S D M B A TPA PROMOTION a
DMBA derivatives Control (only DMBA initiation)e Control (only TFA promotlon) f DMBA 1-OHDMBA 2-OHDMBA 3-OHDMBA 4-OHDMBA 5-OHDMBA 9-OHDMBA 10-OHDMBA DMBA 5,6-dio] DMBA 8,9-diol DMBA 8,9-diol10,11-epoxide I-CH3-DMBA 2-CH3-DMBA • 5-CH3-DMBA 1-fl-DldBA 2-fl-DMBA 5-fl-DMBA 11-~!-DMBA
No. of mice b
Papillomas/ mouse c
30
0
29 28 29 ~0 30 28 29 30 30 29 29
0.10 9.10 0.10 0.07 0.14 0.17 0.I0 0.40 0.47 0.10 0.21
6 100 10 7 14 14 I0 24 30 10 18
28 30 30 29 30 29 28 29
0.30 0.03 0.10 0.40 0.03 0.10 0.20 8.20
20 3 7 34
DERIVATIVES AFTER
Mice with tumors(%)d 0
3
10 15 100
aDIV~BA and derivatives were applied at a dose of 200 nmol and were followed 1 week later by twice weekly applications of 10 ~g of TFA. Sin,riving at ~he 30th week after promotion. CToml number papillonms divided by total number of surviving mice. dpercentage of surviving mice with tumors. eThese mice were only initiated with 200 nmol of DMBA. fThese mice were only promoted twice weekly with 10 #g of TPA for 30 weeks.
O b t a i n i n g d i r e c t e v i d e n c e for d e t e r m i n i n g t h e i m p o r t a n c e o f t h e "bay region~ d i o l - e p o x i d e o f D M B A is difficult b e c a u s e o f t h e h i n d r a n c e o f t h e side c h a i n methyl groups when trying to synthesize them. However, the influence on c a r c i n o g e n ~ c i t y o f t h e a d d i t i o n o f m e t h y l a n d f l u o r o groups in t h e ' b a y r e g i o n ' w o u l d p r o v i d e i n d i r e c t e ~ d e n c e . A m e t h y l or f l u o r o a d d i t i o n t o 1,2 or 5 p o s i t i o n s a l m o s t c o m p l e t e l y b l o c k e d t h e skin t u m o r i n i t i a t i n g a n d V 7 9 m u t a g e n i c activities o f D M B A , whereas f l u o r o a d d i t i o n t o p o s i t i o n 11 d i d n o t . T h e results o f t h i s investigation a n d o t h e r s ~ecently r e p o r t e d [ 4 ; 7 , 2 0 , 3 2 ] i n d i c a t e t h a t a ' b a y r e g i o n ' d i o l - e p o x i d e o f D M B A m a y r e p r e s e n t t h e carcinogerdc a n d m u t a g e n i c f o r m s o f D M B A . T h i s is i n a g r e e m e n t w i t h t h e t h e o r y
217 TABLE 2 MUTAGENICITY OF VARIOUS DMBA DERIVATIVES IN V79 CELLS CO,CULT][VATED WITH NORMAL HAMSTEIt CELLS IN THE CELL-MEDIATED ASSAYa Hydrocarbonb
Cloning efficiency (%)
No. o f ouabain resistant mutant~/10 ~ survivors
None DMBA 2-OHDMBA 3-OHDMBA 4-OHDMBA 5-OHDMBA DMBA-5,6-diol DMBA-8,9"diol c DMBA-S,9"diol-10,11 -oxide 1-CH~-DME~A 2-CH3DMBA 5-CH~-DMBA 1-fI-DMBA 2-fl-DMBA 5-fl-DMBA 11-fl-DMBA
76 18 72 77 69 71 76 70 70 72 73 68 80 67 59 8
0.7 52.0 1.2 1.1 1.2 0.9 1.1
3.2 1.4 0.9 0.9 1.7 1.0 0.9 2.0 37.0
aDMBA and its derivatives wereadded 5 h after 3 × 105 V79 cells were seeded on 2 × 10 ~ irradiated embryonic Golden hamster cells. Two days after treatment, the cells were dissociated and seeded to determine the cloning efficiency of the V79 cells and the frequency of ouabaln res/stant mutants. At the time when the ceRs were seeded for cloning efficiency and ~election of mutation, there were in the D~BA and 11-fi-DMBA treated cultures 1.2--1.6 × 106 V79 cells/Petri dish. In all other c~ses there were 2.1--3.0 X 106 V79 cells/petri dish. bCultures were treated with 4 ~M of the different hy~lrocarbons with the exception of DMBA ~nd 11-fl-DMBA which were treated at the final concentration of 0.1 #M. CThe DMBA-8,9-dio], 1-fl-DMBA and 5-fl-DMBA induced up to 10-fold the mutation frequency at doses higher than 10 pM. The DMBA-8,9-diol-10-oxide was mutagenic when tested in the direct assay at concentrations higher than 10 #M.
p r o p o s e d b y J e r i n a a n d c o - w o r k e r s , w h i c h p r e d i c t s t h a t d i o l . e p o x i d e s in t h e ' b a y r e g i o n ' are t h e principal d e t e r m i n a n t s o f P A H c a r c i n o g e n i c i t y [ 1 0 , 1 1 ] . A l s o o f i m p o r t a n c e is b o t h t h e q u a n t i t a t i v e and q u a l i t a t i v e c o r r e l a t i o n n o t e d b e t w e e n t h e t u m o r - i n i t i a t i n g a c t i v i t y o f D M B A a n d t h e various derivatives i n m o u s e s k i n a n d t h e i r m u t a g e n i c a c t i v i t y in V 7 9 cells. REFERENCES 1 Chouroulinkov, I., Gentil, A., Grover, P.L. and Sims, P. (1976) Tumourqnitiating activities on mouse skin of dihydrodiols derived from benzo(~)pyrene Br. J. Cancer, 34, 523--532. 2 Chouroulinkov L, Gentil, A., Tierney, B., Grover, P. and Sims, F. (1977) The metabolic activation o f 7-methylbenz(o)anthracene in mouse skin: High tumor-initiating activity of the 3,4-dihydrvdiol. Cancer Letters, 3, 247--253.
218
3 Daudel, P., Duquesne, M., Vigny, P., Grocer, P.L. and Sims, P. (1975) Fluorescence spectral evk]ience that benzo(a)pyrene-DNA products in mouse skin arise from diolepoxides. FEBS Lett., 57, 250---253. 4 Dipple, A. and Nebzydoski, J.A. (1978) Evidence for the involvement of a diol-epoxide in the binding of 7,12-dimethylbenz(a)antl~aeene to DNA in cells in culture. Chem.Biol. Interact., 20, 17--26. 5 Fu, P.P. and Harvey, R.G. (1977) Synthesis of the diols and diol epoxides of carcinogenic hydrocarbons. Tetrahed~on Left., 2059--2062. 6 Hubarman, E., Sachs, L.,Yang, S.K. and Gelboin, H.V. (1976) Identification of routsgenie metabolites of ban:~o(a)pyrene in mamrnalian cells. Pros. Natl. Acad. Sci. USA 73, 607--611. 7 Huberman, E. and Slaga~ T,J. (2978) Mutageuicity and tumor-initiating activity of fluorinated 7,12-dimethyl-benz(u)anthracenes. Cancer Res., (In press). S Huberman, E. Yang, S.K., McCourt, D.W. and Gelboin, H.V. (1977) Mutagenicity to mammalian cells in culture by (÷) and (--) t~nns.7,8-dihydroxy-7,8-dihydrobanzo(a)pyrenes and the hydrolysis and reduction products of two stereoisomeric banzo(a)pyrene 7,8-diol-9,10-epoxides. Cancer Letters, 4, 35--43. 9 Hubarman, E. and Sachs, L. (1976) Mutability of different genetic loci in mammalian cells by metabolically activated polycyelic hydrocarbons~ Proc. Nat~. Aead. Sei. USA 73, 88--292. 20 Jarina, D.M. and Daly, J.W. Oxidation at carbon. (1977) In: Drug Metabolism, pp. 15--33. EditorB: D.V. Parke and R.L. Smith. Taylor & Francis, Ltd., London. 12 Jerina, D.M., Lehr, R.E., Yagi, H., Hernandez, O., Dansette, P.M., Wislocki, P.G., Wood, A.W., Change, R.L., Levin, W. and Conney, A.H. (1977) Mutagenicity of benzo(a)pyrane derivatives and the description of a quantum mechanical model which predicts the ease of carbonium ion formation from dlol epoxides. In: In vitro Metabolic Activation and Mutagcnesis Testing, pp. 159--177. Editor~: F.J. de Serres, J.R. Fouts, J.R. Bend and R.M. Philpot. Elsevier/North Holland Biomedical Press, Amsterdam. ]2 Kapitulnik, J., Levin, W,, Conney, A.H., Yagi, H. and Jarina, D.M. (1977) Benzo(a)pyrane-7,8-dihydrodiol ~ more carcinogenic ~han benzo(a)pyrene in newborn mice. Nature, 266, 378--380. 13 King, H.W.S., Osborne, M.R., Belsnd, F . A , harvey, R.G. and Brooks, P~ (2976) (~)7~,8~-dihydroxy-9~,lO~-epoxy.7,8,9,10.tetrahydrobanzo(a)pyrene is an intermediate in the metabolism and binding to DNA of henzo(a)pyrene. Froc. Natl. Acad. Sci. USA 73, 2679--2682. 14 Levin, W., Wood, A.W., Chang, R.L., Slaga, T.J., Yagi, H., Jerina, D.M. and Conney, A.H. (1977) Marked diffsrenees in the tumor-initiating activity of optically pure (~-)and (-)-7,8-dihydroxy-7,8-dihydrn-benzo(o)pyrenein mouse skin. Cancer Res., 37, 2721--2725. 15 Levin, W., Wood, A.W., lragi, H., Dan~ette, P.M., Jerina, D.M. and Conney, A.H. (1976) CarcinugenieJty of benzo(u)pyrene 4,5-7,8-, and 9,10-oxides on meuse skin. Prnc. Nat~. Acad. Sei. USA, 73, 243--247. 16 Levin, W., Wood, A.W., ~agi, H , Jsrina, D.N. and Conney, A.H. (1976) ~-trans-7,8dihydrnxy-7,8.dihydrobcnzo(a)pyrene: A potent skin carcinogen when applied topieall~ to m~ee. Pros. Natl. Acad. Sci. USA, 73, 3867--3871. ~7 Malaveille, C., Bartseh, H.. Grocer, P.L. and Sims, P. {1975) Mutagenicity of non-Kregion diols and din| epoxides of benzo(n)a~thracene and benzo(~lpyrene in S. typhi. murium TA100. Biochem~ Biophys. Res. Corr,'.mun., 66, 693--700. 18 Meehan, T., Stmub, K. and Calvin, M. (1976) Elucidation of hydrocarbon structure in an enzyme-catalyzed benzo(u)pyrene-poly(G) covalent complex. Proc. Natl. Acad. Sci. USA, 73, 1437--1441. 19 ~/Ioore, P.D., Koreeda, M., Wislocki, P.G., Levin, W., Conney, A.H., Yagi, H. and Jerina, D.M. In vitro re~etlons of the diastereomeric 9,10-epoxides of (+) and (--)-7,8-dihydroxy7,8-dihydrobenze(a)pyrene with polyguanylie acid and evidence for formation of an
219
20 21 22
23 24 25 26
27 28
29 30
31 32
33
34
35
enantiomer of each diastereomeric 9,10-epoxide from benzo(a)pyrene in mouse skin. In: Concepts in Drug Metabolism; Editor: D.M. Jerina. American Chemical Society, Washington, D.C. (In press). Moschel, R.C., Baixd, W.M. and Dipple, A, (1977) Metabolic activation of the carcinogen 7,12-dimethylbenz(a)anthracene for D N A binding. Biochem. Biophys. Res. Commun., 76, 1092--1098. Newbold, R.F. and Brooks, P. (1976) Exceptional mutagenicity o~ a benzo(a)pyrene diol epoxide in ct'~Ituredmammalian cells.Nature, 261, 52--54. Osborne, M.R., Thompson, M.H., Tarmy, E.M., Beland, F.A., Harvey, R.G. and Brooks, P. (1976) The re~tctionof 7,8-dihydro-7,8"dihydr)xybenzo(a)-pyrene'9,10"°xide with D N A in relation to the benzo(a)pyrene D N A praducts isolated from cells.Chem.-Biol. I~teract, 13,343--348. Slaga,T.J.,Viaje, A., Bracken, W.M., Berry, D.L., Fischer, S.M., Miller,D.R. and LeClerc, S.M. (1977) Skin-tumor initiatingability of benzo(a)pyrene-7,8-diol'9,10"epoxide (anti) when applied topically in tetrahydrofuran. Cancer Letters, 3, 23--30. Slaga, T.J., Huberman, E., Selkirk,J.K., Harvey, R.G. and Bracken, W.M. (1978) Carcinogenicity and rnutagenicity of benz(a)anthrac,.me diols and diol-epoxides. Cancer Res., 38~ 1699--1704. Slaga, T.J., Bracken, W.M., Viaje, A., Levin, W., Yagi, H., Jerina, D.M. and Conney, A.H. (1977) Comparison of the tumor-initiating activitiesof benzo(a)pyrene arene oxides and diol-ejzoxides.Cancer Res., 37, 4130--4133. Slaga, T.J., Vi~de, A., Berry, D.L., Bracken, W., Buty, S.G. and Scribner, J.D. (1976) Skln-tumor initiatingability of benzo(a}pyrene-4,5-7,8- and 7,8-diol-9,10-epoxides and 7,8-dioi. C'.an~er Letters, 2, 115--122. Sims, P., Grover, P.L., Swais|and, A., Pal, K. and Hewer, A. (1974) Metabolic activation of benzo(a)pyrene proceeds by a diol-epoxide. Nature, 252, 326--328. Thakker, D.R., Yagi, H., Akagi, H., Koreeda, M., Lu, A.Y.H., Levin, W., Wood, A.W., Conney, A.H. and Jerina, D.M. (t977) Metabolism of henzo(a)pyrene. VI. Stereoselective metabolism of benzo(a)pyrene and benzo(a)pyrene-7,8-dihydrodiol ~o diol epoxides. Chem.-Biol. Interact. 16, 281--300. Weinstein, I.B., Jeffrey, A.M., Jennette, K.W., Blob~tein, S.H., Harvey, R.G., Harris, C., Autrup, H., Kasaii, H. and Nakanishi, K. (1976) Benzo(a)pyrene diol epoxides as intermediates in nucleic acid binding in vitro and in vivo, Science, 193, 592--595. Wislocki, P.G., Wood, A.W., Chang, R.L., Levin, W., Yagi, H., Hernandez, O., Dansettc, P.M., Jerina, D.M. and Conney, A.H. (1976) Mutagenicity and cytotoxicity of benzo(a)pyrene oxides, phenols, quinones and dihydrodiols in bacterial and mammalian cells. Cancer Res., 36, 3350--3357. Wislocki, P.G., Wood, A.W., Chang, R.L., Levin, W., Yagi, H., Hernandez, O., Jerina, D.M. and Conney, A.H. (1976) High mutagenicity and toxicity of a diol epoxide derived from benzo(a)]~y:ene. Biochem. Biophys. Res. Commun., 68, 1006--1012. Wood, A.W., Chang, R.L., Levin, W., Lehr, R,E., Sehoefer-Reddel:!, M., Kaxle, J.M., Jerina, D.M. a~ad Conney, A.H. ~1977) Mutagenicity and cytotoxicity of benz(a)anthracene diol epoxides and tetrahydroepoxides: exceptional activity of the bay region 1,2-epoxides. Proc. Natl. Acad. Sci. USA, 74, 2746--2750. Wood, A.W., Levin, W.~ Chang, R.L., Lehr, R.E., Schaefer-Redder, M., Karle, J.M., Jerina, D.M. and Cooney, A.H. (1977) Tumorigenicity of five dihydrodiols of benz(a) anthracene on mouse skin: exceptional activity of benz(a)anthracene-3,4-diol. Proc. Natl. Acad. Sci. USA, 74, 3176--3179. Wood, A.W., Levin, W., Lu, A.Y,H., Ryan, D., West, S.B., Lehr, R.E., Schaefer-Redder, M., Jerina, D.M. and Co:oney, A.H. (1976) Mutagenicit)' of metabolically activated benz(a}anthracene-3,4*dihydrodiol. Evidence for bay region activation of carcinogenic polycyclic hydrocarbon!;. Biochem. Biophys. Res. Commun., 72, 680--686. Wood, A.W, Levin,W., ~yan, D., Thomas, P.E., Yagi, H., Moh, H.D., Thakker, D.R., Jerina, D.M. and Conney, A.H. (1977) High Mutagenicity of metabolically activated
220 chryscne-l,2-dihydrodiot. Evidence for bay region activation of chrysene. Biochem. Biopbys. Res. Commun., 78,847--854. 36 Wood, A.W., Levin, W., Thomas, P.E., Ryan, D., tL~rlc, J.M., Yagi, H., Jerina, D.M. and Conney, A.H. (1978) Metabolic activation of dibcnzo(a,h)anthracene i~nd its dihydrodiols to bacterial mutagens. Cancer Res., 38, 1967--1973. 37 Yang, S.K., McCourt, D.W,, Roller, P.P. and Gelboin, H.V. (1976) Enzymatic conversion o f benzo(a)pyrene leading predominantly to the diol-epoxide-r-7,t-8.dihydroxy~t-9, 10-oxy-7,8,9,10-tetrahydrobenzo(a)pyrene titrough a single enantiomer of 4-7,t-Sdihydroxy-7,8-dihydrobenzo(a)pyrene. Proc. Natl. Acad. Sci. USA, 73, 2594--2598.
213
THE I M P O R T A N C E O F THE " B A Y R E G I O N " D I O L - E P O X I D E I N 7,12D I M E T H Y L B E N Z | a ] 2 ~ N T H R A C E N E S K I N TUMO]~ I N I T I A T I O N A N D MUTAGENESIS*,** T.J. SLAGAt, E. HUBERMAN, J. DiGIOVANNIt t and G. GLEASON Biology Division, Oak Ridge National Laboratory, Oak Ridge, Tennessee 37830 (U.&A.) R.G. H A R V E Y Ben May Laboratory for Cancer Research, University o f Chicago, Chicago, Illinois 60637
(U.S.A.) (Received 5 October 1978) (Revised version received 22 November 195'8) (Accepted 5 December 1978)
SUMMARY T h e skin tumor-initiating and V 7 9 m u t a g e n i c activities of various deriw'~tives o f 7 , 1 2 - d i m e t h y l b e n z [ a ] a n t h r a c e n e (DMBA) were investigated to d e t e r m i n e w h a t possible cellular metabolite(s) m a y be responsible for its carcinogenivity a n d / o r mutagenict~y. 1-,2-,3.,4- and 5 - h y d r o x y D M B A were f o u n d t o be essentially inactive as skin t u m o r initiators whereas 9- and 1 0 - h y d r o x y D M B A h a d w e a k activity. The (±)-trans D M B A 8,9- and 5,6-dihydrodiols were also *Research sponsored jointly by the NIH under Interagcney Agreement Grant CA-20076 and by the U.S. Department of Energy, under contract W-7405-eng-26 with the Union Carbide Corporation. **After our manuscript was submitted for publication, Maleveille and coworkers reported the high mutagenie activity of the 3,4-dihydrodiol of DMBA in S. typhimurium TA100 in the presence of a rat liver metabolizing system which agrees with the data presented here. See, Malaveflle, C. Bartseh, H., Tierney, B., Grovar, P.L. and Sims, P. (1978). Miarosome~ mediated mutagenicities of the dihydrodio]s of 7,12.dirnethyihenz[a]anthraeene: High mutagenic activity of the 3,4-dihydrodiol. Bioehem. Biophys. Res. Commun., 83, 1468--1473. t To whom requests for reprints and correspondence shound be addrea~ed to: Biology Division, Oak Ridge National Laboratory, Post Office Box Y, Oak Ridge, Tennessee 37830, U.S.A. T1"Present add~.e~: McPadie Laboratory for Cancer Reseaxeh, University of Wisconsin Medical SchooL, Madison, Wisconsin, U.S.A. Abbreviations: BP, benzo[a]pyrene; DMBA, 7,12-dimethylbenz[a]anthracene; PAH, polycyclic aromatic hydrocarbons; 1-. 2-, 3-, 4-, 5-, 9- and 10-OH DMBA, various phenols of DMBA; DMBA 5,6-diol, (±)trans-5,6-dihydroxy~5,6-d~hydro.DMBA; DMBA 8,9-diol, (±)traas8,9-dihydroxy-8,9-dihydro-DMBA; DMBA 8,9-diol-.~.0,11-epoxlde, (±)trans-8~, 9~-dihydroxyI0~,11~-epoxy-8,9,10,11-tetrahydro-DMBA; 1-CH~-, 2-CH3-, 2-CH~, and 5-CH3-DMBA, either 1-, 2 or 5,7,12-trimethylbenz[a ]anthracene~ 1-, 2-, 5-, and 11-fl-DMBA, 1-, 2-, 5- and 11-flaoro-DMBA; TPA, 12-O-tetradecanoyi-phorbr~l- 13-acetate.
214
essentially inactiveas skin tumor initiatorsand (-+)-DMBA 8~,9~-diol-!0a,llaepoxide had weak skin tumor initiatingactivity.All of the above tested derivatives of D M B A were essentiallyinactive as mutagens in the cell-mediated or direct V 7 9 mutagenesis systems. A methyl or fluoro addition to the I, 2 or 5 positions almost completely blocked the skin tumor initiatingand V 7 9 mutagenic activitiesof DIVIBA, whereas a fluoro addition to position 11 did not. F r o m our da'~awe suggest that a 'bay region' diol-epoxide m a y be important in D M B A carcinogenicity and mutagenicity.
INTRODUCTION Recent evidence has revealed that 'bay region' diol-epoxides of benzo[a]pyrene (BP), benz[a]anthracene, chrysene, 7-methylbenz[a]anthracene and dibenz[a,h]anthracene are highly carcinogenic and mutagenie [1,2,6,8,10--12, 14--17,21,23--26,30--36]. Furthermore, in vitro and in vivo binding [3,13,18, 19,22,27,29] and metabolism [28,37] studies, as well as the above mu~agenesis and carcinogenesis studies, have led to the conclusion that a 'bay region' diolepoxide of BP is responsible for its c~_reinogenicity and mutagenicity. Since DMBA is one of the most potent carcinogenic PAH, it would be of interest to determine whether DMBA is also activated into a carcinogen and mut~gen via this pathway. Moschel et ~L. [20] have recently presented fluorescence data which implicates the 'bay region' diol.epoxide of DMBA in the interaction of DMBA with cellular DNA. In addition, binding of DMBA to cells in culture analyzed by LH20 chromatot,~aphy also suggests that a 'bay region' diol epoxide is involved in DMBA binding [3]. In this study we compared the skin tumor initiation in mice and mutagenesis in V79 cells of a 'non-bay region' diol epoxide, several diols, phenols, and methyl and fluoro derivatives of DMBA. The data suggest that a "bay region' diol epoxide may be important in DMBA carcinogenicity and mutagenicity. A 'bay region' occurs in a polycyclic hydrocarbon when an angularly ,~used benzo ring is present [!0,11]. MATERIALS AND METHODS Chemicals DMBA was purchased from Sigma Chemical Company, St. Louis, Missouri and was more than 99% pure. 12-O-tetradecanoyl-plhorbol.13-acetate (TPA) was obtained from Dr. P. Borcher~, Universil~y of Minnesota, Minneapolis, Minnesota. The DMBA diols and diol-epoxide were synthesized and purified as previously described [5]. The 1-, 2-, 5-, and ll-fI-DMBA, the 1-, 2-, and 5CH3DMBA and the 1-, 2-, 3-, 4-, 5-, 9-, and 10-OH DMBA were generous gifts from Professor M. Newman, Ohio State University, Columbus, Ohio. All o:[ the hydrocarbons were prepared in the dark or in yellow light immediately before use.
215 Tumor experiments Female CD1 mice were purchased from Charles River Farms, North Wilmington, Massachusetts. Mice, 7--9 weeks old, were shaved with surgical clippers 2 days before treatment and only those in the resting phase of the hair cycle were used. in the tumor experiments groups of 30 animals received a single topical application of the test compound, followed I week later by twice weekly applications of TPA. The incidence of papillomas was recorded weekly and they were removed at random for histological verific~.tion. The DMBA phenols, diols and fluoro and CH3 derivatives were applied j.opically at a dose of 200 nmol in acetone whereas the diol-epoxide of DMBA was applied topically at a dose of 200 nmol in tetrahydrofuran. Direct and cell-mediated mu tagenesis assay Ouabain resistance was tested by seeding 3 × 10 s V79 ceils in 4 ml of medium into a Petri dish without the hamster ceils for the direct assay [8], or on a 1-day-old monolayer of 5000 F~-irradiated normal golden hamster cells seeded at 2 × 106 cells/Petri dish for the cell-mediated assay [6,9]. The cells were cultured as previously described [9]. DMBA and derivatives were added 5 h after the V79 cells were seeded in 1 ml of medium. Incubated for 2 days and then dissociated with a trypsin-EDTA solution and seeded at 200 cells/Petri dish in 5 ml of medium to detet~fine cloning efficiency and at 10 s cells/Petri dish in 4 ml of medium to determine the frequency of ouabain-resistant mutants. For selection of the mutants, ouabain at a final concentration of 1 mM was added in 1 ml of medium 2 days after cell seeding, The colonies were counted after staining with Giemsa. Cloning efficiency was determined by counting the number of colonies in 6--8 Petri dishes/point at 7--8 days after cell seeding, and the frequency of ouabain-resistant mutants was determined by counting 24--32 Petri dishes]point at 14--16 days after cell seeding. The results were based on 2 experiments/point. The number of mutants after induction with DMBA or l l - f l - D M B A varied up to 30~d. In all other cases, the number of mutants in the different experiments varied up to 60%. RESULTS A N D DISCUSSION
Table 1 shows the relative skin tumor-initiating activities of the various derivatives of DMBA. The 1-, 2-, 3-, 4 and 5-OH DMBA were found not to have skin tumor-initiating activity whereas the 9- and 10-OH DMBA had weak activity. The 5,6- and 8,9-diols of DMBA were also found to be inactive and the (±)DMBA 88, 9~-diol-10a, ll~-epoxide had only weak skin tumor-initiating activity. It is interesting to point out that, as in the case of the tmnor experiments, DMBA and its ll-fluors derivatives were potent mutagens when tested in the cell mediated assay whii~e all other derivatives were either inactive or exhibited poor activity (Table I!). In the direct V79 mutagenesis assay in which there is no detectable polycyclk~ hydrocarbon metabolism neither DMBA nor any of the tested phenols, diols and diol-epoxide were active as mutagens at hydrocarbon concentration of up to 4 ~M.
216 TABLE 1 S K I N T U M O R INITIATING ACTIVITIES OF. V A R I O U S D M B A TPA PROMOTION a
DMBA derivatives Control (only DMBA initiation)e Control (only TFA promotlon) f DMBA 1-OHDMBA 2-OHDMBA 3-OHDMBA 4-OHDMBA 5-OHDMBA 9-OHDMBA 10-OHDMBA DMBA 5,6-dio] DMBA 8,9-diol DMBA 8,9-diol10,11-epoxide I-CH3-DMBA 2-CH3-DMBA • 5-CH3-DMBA 1-fl-DldBA 2-fl-DMBA 5-fl-DMBA 11-~!-DMBA
No. of mice b
Papillomas/ mouse c
30
0
29 28 29 ~0 30 28 29 30 30 29 29
0.10 9.10 0.10 0.07 0.14 0.17 0.I0 0.40 0.47 0.10 0.21
6 100 10 7 14 14 I0 24 30 10 18
28 30 30 29 30 29 28 29
0.30 0.03 0.10 0.40 0.03 0.10 0.20 8.20
20 3 7 34
DERIVATIVES AFTER
Mice with tumors(%)d 0
3
10 15 100
aDIV~BA and derivatives were applied at a dose of 200 nmol and were followed 1 week later by twice weekly applications of 10 ~g of TFA. Sin,riving at ~he 30th week after promotion. CToml number papillonms divided by total number of surviving mice. dpercentage of surviving mice with tumors. eThese mice were only initiated with 200 nmol of DMBA. fThese mice were only promoted twice weekly with 10 #g of TPA for 30 weeks.
O b t a i n i n g d i r e c t e v i d e n c e for d e t e r m i n i n g t h e i m p o r t a n c e o f t h e "bay region~ d i o l - e p o x i d e o f D M B A is difficult b e c a u s e o f t h e h i n d r a n c e o f t h e side c h a i n methyl groups when trying to synthesize them. However, the influence on c a r c i n o g e n ~ c i t y o f t h e a d d i t i o n o f m e t h y l a n d f l u o r o groups in t h e ' b a y r e g i o n ' w o u l d p r o v i d e i n d i r e c t e ~ d e n c e . A m e t h y l or f l u o r o a d d i t i o n t o 1,2 or 5 p o s i t i o n s a l m o s t c o m p l e t e l y b l o c k e d t h e skin t u m o r i n i t i a t i n g a n d V 7 9 m u t a g e n i c activities o f D M B A , whereas f l u o r o a d d i t i o n t o p o s i t i o n 11 d i d n o t . T h e results o f t h i s investigation a n d o t h e r s ~ecently r e p o r t e d [ 4 ; 7 , 2 0 , 3 2 ] i n d i c a t e t h a t a ' b a y r e g i o n ' d i o l - e p o x i d e o f D M B A m a y r e p r e s e n t t h e carcinogerdc a n d m u t a g e n i c f o r m s o f D M B A . T h i s is i n a g r e e m e n t w i t h t h e t h e o r y
217 TABLE 2 MUTAGENICITY OF VARIOUS DMBA DERIVATIVES IN V79 CELLS CO,CULT][VATED WITH NORMAL HAMSTEIt CELLS IN THE CELL-MEDIATED ASSAYa Hydrocarbonb
Cloning efficiency (%)
No. o f ouabain resistant mutant~/10 ~ survivors
None DMBA 2-OHDMBA 3-OHDMBA 4-OHDMBA 5-OHDMBA DMBA-5,6-diol DMBA-8,9"diol c DMBA-S,9"diol-10,11 -oxide 1-CH~-DME~A 2-CH3DMBA 5-CH~-DMBA 1-fI-DMBA 2-fl-DMBA 5-fl-DMBA 11-fl-DMBA
76 18 72 77 69 71 76 70 70 72 73 68 80 67 59 8
0.7 52.0 1.2 1.1 1.2 0.9 1.1
3.2 1.4 0.9 0.9 1.7 1.0 0.9 2.0 37.0
aDMBA and its derivatives wereadded 5 h after 3 × 105 V79 cells were seeded on 2 × 10 ~ irradiated embryonic Golden hamster cells. Two days after treatment, the cells were dissociated and seeded to determine the cloning efficiency of the V79 cells and the frequency of ouabaln res/stant mutants. At the time when the ceRs were seeded for cloning efficiency and ~election of mutation, there were in the D~BA and 11-fi-DMBA treated cultures 1.2--1.6 × 106 V79 cells/Petri dish. In all other c~ses there were 2.1--3.0 X 106 V79 cells/petri dish. bCultures were treated with 4 ~M of the different hy~lrocarbons with the exception of DMBA ~nd 11-fl-DMBA which were treated at the final concentration of 0.1 #M. CThe DMBA-8,9-dio], 1-fl-DMBA and 5-fl-DMBA induced up to 10-fold the mutation frequency at doses higher than 10 pM. The DMBA-8,9-diol-10-oxide was mutagenic when tested in the direct assay at concentrations higher than 10 #M.
p r o p o s e d b y J e r i n a a n d c o - w o r k e r s , w h i c h p r e d i c t s t h a t d i o l . e p o x i d e s in t h e ' b a y r e g i o n ' are t h e principal d e t e r m i n a n t s o f P A H c a r c i n o g e n i c i t y [ 1 0 , 1 1 ] . A l s o o f i m p o r t a n c e is b o t h t h e q u a n t i t a t i v e and q u a l i t a t i v e c o r r e l a t i o n n o t e d b e t w e e n t h e t u m o r - i n i t i a t i n g a c t i v i t y o f D M B A a n d t h e various derivatives i n m o u s e s k i n a n d t h e i r m u t a g e n i c a c t i v i t y in V 7 9 cells. REFERENCES 1 Chouroulinkov, I., Gentil, A., Grover, P.L. and Sims, P. (1976) Tumourqnitiating activities on mouse skin of dihydrodiols derived from benzo(~)pyrene Br. J. Cancer, 34, 523--532. 2 Chouroulinkov L, Gentil, A., Tierney, B., Grover, P. and Sims, F. (1977) The metabolic activation o f 7-methylbenz(o)anthracene in mouse skin: High tumor-initiating activity of the 3,4-dihydrvdiol. Cancer Letters, 3, 247--253.
218
3 Daudel, P., Duquesne, M., Vigny, P., Grocer, P.L. and Sims, P. (1975) Fluorescence spectral evk]ience that benzo(a)pyrene-DNA products in mouse skin arise from diolepoxides. FEBS Lett., 57, 250---253. 4 Dipple, A. and Nebzydoski, J.A. (1978) Evidence for the involvement of a diol-epoxide in the binding of 7,12-dimethylbenz(a)antl~aeene to DNA in cells in culture. Chem.Biol. Interact., 20, 17--26. 5 Fu, P.P. and Harvey, R.G. (1977) Synthesis of the diols and diol epoxides of carcinogenic hydrocarbons. Tetrahed~on Left., 2059--2062. 6 Hubarman, E., Sachs, L.,Yang, S.K. and Gelboin, H.V. (1976) Identification of routsgenie metabolites of ban:~o(a)pyrene in mamrnalian cells. Pros. Natl. Acad. Sci. USA 73, 607--611. 7 Huberman, E. and Slaga~ T,J. (2978) Mutageuicity and tumor-initiating activity of fluorinated 7,12-dimethyl-benz(u)anthracenes. Cancer Res., (In press). S Huberman, E. Yang, S.K., McCourt, D.W. and Gelboin, H.V. (1977) Mutagenicity to mammalian cells in culture by (÷) and (--) t~nns.7,8-dihydroxy-7,8-dihydrobanzo(a)pyrenes and the hydrolysis and reduction products of two stereoisomeric banzo(a)pyrene 7,8-diol-9,10-epoxides. Cancer Letters, 4, 35--43. 9 Hubarman, E. and Sachs, L. (1976) Mutability of different genetic loci in mammalian cells by metabolically activated polycyelic hydrocarbons~ Proc. Nat~. Aead. Sei. USA 73, 88--292. 20 Jarina, D.M. and Daly, J.W. Oxidation at carbon. (1977) In: Drug Metabolism, pp. 15--33. EditorB: D.V. Parke and R.L. Smith. Taylor & Francis, Ltd., London. 12 Jerina, D.M., Lehr, R.E., Yagi, H., Hernandez, O., Dansette, P.M., Wislocki, P.G., Wood, A.W., Change, R.L., Levin, W. and Conney, A.H. (1977) Mutagenicity of benzo(a)pyrane derivatives and the description of a quantum mechanical model which predicts the ease of carbonium ion formation from dlol epoxides. In: In vitro Metabolic Activation and Mutagcnesis Testing, pp. 159--177. Editor~: F.J. de Serres, J.R. Fouts, J.R. Bend and R.M. Philpot. Elsevier/North Holland Biomedical Press, Amsterdam. ]2 Kapitulnik, J., Levin, W,, Conney, A.H., Yagi, H. and Jarina, D.M. (1977) Benzo(a)pyrane-7,8-dihydrodiol ~ more carcinogenic ~han benzo(a)pyrene in newborn mice. Nature, 266, 378--380. 13 King, H.W.S., Osborne, M.R., Belsnd, F . A , harvey, R.G. and Brooks, P~ (2976) (~)7~,8~-dihydroxy-9~,lO~-epoxy.7,8,9,10.tetrahydrobanzo(a)pyrene is an intermediate in the metabolism and binding to DNA of henzo(a)pyrene. Froc. Natl. Acad. Sci. USA 73, 2679--2682. 14 Levin, W., Wood, A.W., Chang, R.L., Slaga, T.J., Yagi, H., Jerina, D.M. and Conney, A.H. (1977) Marked diffsrenees in the tumor-initiating activity of optically pure (~-)and (-)-7,8-dihydroxy-7,8-dihydrn-benzo(o)pyrenein mouse skin. Cancer Res., 37, 2721--2725. 15 Levin, W., Wood, A.W., lragi, H., Dan~ette, P.M., Jerina, D.M. and Conney, A.H. (1976) CarcinugenieJty of benzo(u)pyrene 4,5-7,8-, and 9,10-oxides on meuse skin. Prnc. Nat~. Acad. Sei. USA, 73, 243--247. 16 Levin, W., Wood, A.W., ~agi, H , Jsrina, D.N. and Conney, A.H. (1976) ~-trans-7,8dihydrnxy-7,8.dihydrobcnzo(a)pyrene: A potent skin carcinogen when applied topieall~ to m~ee. Pros. Natl. Acad. Sci. USA, 73, 3867--3871. ~7 Malaveille, C., Bartseh, H.. Grocer, P.L. and Sims, P. {1975) Mutagenicity of non-Kregion diols and din| epoxides of benzo(n)a~thracene and benzo(~lpyrene in S. typhi. murium TA100. Biochem~ Biophys. Res. Corr,'.mun., 66, 693--700. 18 Meehan, T., Stmub, K. and Calvin, M. (1976) Elucidation of hydrocarbon structure in an enzyme-catalyzed benzo(u)pyrene-poly(G) covalent complex. Proc. Natl. Acad. Sci. USA, 73, 1437--1441. 19 ~/Ioore, P.D., Koreeda, M., Wislocki, P.G., Levin, W., Conney, A.H., Yagi, H. and Jerina, D.M. In vitro re~etlons of the diastereomeric 9,10-epoxides of (+) and (--)-7,8-dihydroxy7,8-dihydrobenze(a)pyrene with polyguanylie acid and evidence for formation of an
219
20 21 22
23 24 25 26
27 28
29 30
31 32
33
34
35
enantiomer of each diastereomeric 9,10-epoxide from benzo(a)pyrene in mouse skin. In: Concepts in Drug Metabolism; Editor: D.M. Jerina. American Chemical Society, Washington, D.C. (In press). Moschel, R.C., Baixd, W.M. and Dipple, A, (1977) Metabolic activation of the carcinogen 7,12-dimethylbenz(a)anthracene for D N A binding. Biochem. Biophys. Res. Commun., 76, 1092--1098. Newbold, R.F. and Brooks, P. (1976) Exceptional mutagenicity o~ a benzo(a)pyrene diol epoxide in ct'~Ituredmammalian cells.Nature, 261, 52--54. Osborne, M.R., Thompson, M.H., Tarmy, E.M., Beland, F.A., Harvey, R.G. and Brooks, P. (1976) The re~tctionof 7,8-dihydro-7,8"dihydr)xybenzo(a)-pyrene'9,10"°xide with D N A in relation to the benzo(a)pyrene D N A praducts isolated from cells.Chem.-Biol. I~teract, 13,343--348. Slaga,T.J.,Viaje, A., Bracken, W.M., Berry, D.L., Fischer, S.M., Miller,D.R. and LeClerc, S.M. (1977) Skin-tumor initiatingability of benzo(a)pyrene-7,8-diol'9,10"epoxide (anti) when applied topically in tetrahydrofuran. Cancer Letters, 3, 23--30. Slaga, T.J., Huberman, E., Selkirk,J.K., Harvey, R.G. and Bracken, W.M. (1978) Carcinogenicity and rnutagenicity of benz(a)anthrac,.me diols and diol-epoxides. Cancer Res., 38~ 1699--1704. Slaga, T.J., Bracken, W.M., Viaje, A., Levin, W., Yagi, H., Jerina, D.M. and Conney, A.H. (1977) Comparison of the tumor-initiating activitiesof benzo(a)pyrene arene oxides and diol-ejzoxides.Cancer Res., 37, 4130--4133. Slaga, T.J., Vi~de, A., Berry, D.L., Bracken, W., Buty, S.G. and Scribner, J.D. (1976) Skln-tumor initiatingability of benzo(a}pyrene-4,5-7,8- and 7,8-diol-9,10-epoxides and 7,8-dioi. C'.an~er Letters, 2, 115--122. Sims, P., Grover, P.L., Swais|and, A., Pal, K. and Hewer, A. (1974) Metabolic activation of benzo(a)pyrene proceeds by a diol-epoxide. Nature, 252, 326--328. Thakker, D.R., Yagi, H., Akagi, H., Koreeda, M., Lu, A.Y.H., Levin, W., Wood, A.W., Conney, A.H. and Jerina, D.M. (t977) Metabolism of henzo(a)pyrene. VI. Stereoselective metabolism of benzo(a)pyrene and benzo(a)pyrene-7,8-dihydrodiol ~o diol epoxides. Chem.-Biol. Interact. 16, 281--300. Weinstein, I.B., Jeffrey, A.M., Jennette, K.W., Blob~tein, S.H., Harvey, R.G., Harris, C., Autrup, H., Kasaii, H. and Nakanishi, K. (1976) Benzo(a)pyrene diol epoxides as intermediates in nucleic acid binding in vitro and in vivo, Science, 193, 592--595. Wislocki, P.G., Wood, A.W., Chang, R.L., Levin, W., Yagi, H., Hernandez, O., Dansettc, P.M., Jerina, D.M. and Conney, A.H. (1976) Mutagenicity and cytotoxicity of benzo(a)pyrene oxides, phenols, quinones and dihydrodiols in bacterial and mammalian cells. Cancer Res., 36, 3350--3357. Wislocki, P.G., Wood, A.W., Chang, R.L., Levin, W., Yagi, H., Hernandez, O., Jerina, D.M. and Conney, A.H. (1976) High mutagenicity and toxicity of a diol epoxide derived from benzo(a)]~y:ene. Biochem. Biophys. Res. Commun., 68, 1006--1012. Wood, A.W., Chang, R.L., Levin, W., Lehr, R,E., Sehoefer-Reddel:!, M., Kaxle, J.M., Jerina, D.M. a~ad Conney, A.H. ~1977) Mutagenicity and cytotoxicity of benz(a)anthracene diol epoxides and tetrahydroepoxides: exceptional activity of the bay region 1,2-epoxides. Proc. Natl. Acad. Sci. USA, 74, 2746--2750. Wood, A.W., Levin, W.~ Chang, R.L., Lehr, R.E., Schaefer-Redder, M., Karle, J.M., Jerina, D.M. and Cooney, A.H. (1977) Tumorigenicity of five dihydrodiols of benz(a) anthracene on mouse skin: exceptional activity of benz(a)anthracene-3,4-diol. Proc. Natl. Acad. Sci. USA, 74, 3176--3179. Wood, A.W., Levin, W., Lu, A.Y,H., Ryan, D., West, S.B., Lehr, R.E., Schaefer-Redder, M., Jerina, D.M. and Co:oney, A.H. (1976) Mutagenicit)' of metabolically activated benz(a}anthracene-3,4*dihydrodiol. Evidence for bay region activation of carcinogenic polycyclic hydrocarbon!;. Biochem. Biophys. Res. Commun., 72, 680--686. Wood, A.W, Levin,W., ~yan, D., Thomas, P.E., Yagi, H., Moh, H.D., Thakker, D.R., Jerina, D.M. and Conney, A.H. (1977) High Mutagenicity of metabolically activated
220 chryscne-l,2-dihydrodiot. Evidence for bay region activation of chrysene. Biochem. Biopbys. Res. Commun., 78,847--854. 36 Wood, A.W., Levin, W., Thomas, P.E., Ryan, D., tL~rlc, J.M., Yagi, H., Jerina, D.M. and Conney, A.H. (1978) Metabolic activation of dibcnzo(a,h)anthracene i~nd its dihydrodiols to bacterial mutagens. Cancer Res., 38, 1967--1973. 37 Yang, S.K., McCourt, D.W,, Roller, P.P. and Gelboin, H.V. (1976) Enzymatic conversion o f benzo(a)pyrene leading predominantly to the diol-epoxide-r-7,t-8.dihydroxy~t-9, 10-oxy-7,8,9,10-tetrahydrobenzo(a)pyrene titrough a single enantiomer of 4-7,t-Sdihydroxy-7,8-dihydrobenzo(a)pyrene. Proc. Natl. Acad. Sci. USA, 73, 2594--2598.