[CANCER RESEARCH 40, 642-649, March 1980]0008-5472/80/0040-0000$02.00

Mutagenicity and Tumor-initiating Activity of Cyclopenta(c,d)pyrene andStructurally Related Compounds

Alexander W. Wood,1 Wayne Levin, Richard L. Chang, Mou-Tuan Huang, Dene E. Ryan, Paul E. Thomas,Roland E. Lehr,2 Subodh Kumar, Masato Koreeda,3 Hirashi Akagi, Yitzhak lttah, Patrick Dansette, HaruhikoYagi, Donald M. Jerina, and Allan H. Conney

Department of Biochemistry and Drug Metabolism, Hoffmann-LaRoche Inc., Nutley, New Jersey 071 10 fA. W. W., W. L., A. L. C., M-T. H., 0. E. R., P. E. T., A. H.C.); Department of Chemistry, University of Oklahoma, Norman, Oklahoma 73019 (A. E. L., S. K.); Laboratorie de Chimie, Ecole Normale Superieure, 75231 ParisCedex 05, France (P. 0.); Department of Chemistry, University of Michigan, Ann Arbor, Michigan 48109 (M. K., H. A.]; and Laboratory of Bioorganic Chemistry,National Institutes cf Arthritis, Metabolism, and Digestive Diseases, NIH, Bethesda, Maryland 20205 IY. I., H. Y., D. M. J.j

ABSTRACT

The biological activities of benzo(a)pyrene, cycbopenta(c,d)pyrene, and i 2 other structurally related compounds wereassessed by mutagenicity studies with bacterial and mammahan cells and/or skin tumonigenicity studies with mice. Theability of the parent hydrocarbons to be metabolically activatedto mutagenic products was examined in strains TA98 andTAi 00 of Salmonella typhimurium, using 3 experimental protocols. In each case, cycbopenta(c,d)pyrene was metabolicallyactivated to products mutagenic to the bacteria to a greaterextent than was benzo(a)pynene. However, 7,8-dihydrobenzo(a)pyrene and 9, i 0-dihydrobenzo(e)pyrene were thebest substrates for metabolic activation to bacterial mutagens.Highly purified epoxide hydnaseadded to a purified and reconstituted monooxygenase system readily abolished the mutagenic activity observed in strain TA100 of S. typhimurium whencycbopenta(c,d)pyrene was the substrate, but not whenbenzo(a)pyrene was the substrate. Inherent mutagenicity ofseveral epoxides of the hydrocarbons generally paralleled theability of their potential metabolic precursors to be activated tomutagens. 1-Pyrenyboxirane and i 0, i i -dihydrocycboheptapyrene 8,9-oxide were highly mutagenic in strains TA98 andTA100 of S. typhimurium, and in the former strain these activities were comparable to that observed with 9,i 0-epoxy7,8,9,i 0-tetrahydrobenzo(a)pyrene. 4-Pyrenyboxiranewas significantly less mutagenic than was i -pyrenyboxirane in bothstrains of bacteria and in mammalian cells. Benzo(a)pyrenewas over 20 times more turnonigenic than was cycbopenta(c,d)pyrene, and it was the most potent of the i i compoundstested for tumor-initiating activity in 2-stage initiation-promotion experiments on the skin of mice. Cycbopenta(c,d)pyrene had tumor-initiating activity comparable to that of benzo(a)anthracene, but it was significantly less active than chrysene. Thus, contrary to inferences made from its high mutagenic activity, cycbopenta(c,d)pyrene is a weak tumor initiatoron mouse skin.

INTRODUCTION

The polycyclic aromatic hydrocarbon CPP4 (Chart i ) is a

Received August 31, i 979; accepted November 20, i 979.1 To whom requests for reprints should be addressed.

2 Recipient of National Cancer Institute Grant 5-ROl -CA-22985-02, which

provided financial support for part of this study.3 Recipient of NIH Grant C825185, which provided financial support for parts

of this study.4 The abbreviations used are: CPP, cyclopenta(c,d)pyrene; B(a)P,

benzo(a)pyrene; TPA, 12-O-tetradecanoylphorbol-1 3-acetate; 7,8-H2B(a)P,7,8-

widespread environmental contaminant (i 0, i 2, 34), the concentration of which far exceeds that of the ubiquitous carcinogen B(a)P in sources such as automobile exhaust (12). Considerable attention has been drawn to CPP because of itspresence in the environment, its carcinogenicity upon s.c.injection into mice (27), and the fact that it is metabolicallyactivated by liver microsornal preparations to products highlymutagenic toward strains of S. typhimurium (6). The currentinterest in CPP is exemplified by the fact that 4 syntheses ofthe hydrocarbon have been described recently (i i , i 3, i 8,28), despite the fact that the batter 2 (i 8, 28) are essentiallyidentical to the initial preparation (i i , i 3). Although Eisenstadtand Gold (6) have suggested that CPP should be highly tumorigenic based on its mutagenicity in S. typhimurium, the onlyexisting tumor study (27) on the hydrocarbon offers little opportunity to compare its activity to that of other carcinogenicpolycyclic aromatic hydrocarbons. Thus, the present study wasdesigned to provide information on those factors responsiblefor the mutagenicity of CPP upon metabolic activation and torelate the tumor-initiating activity of CPP to that of other carcinogenic hydrocarbons.

MATERIALS AND METHODS

Materials.Arocbon1254 (LotKCi 2-638) wasobtainedfromMonsanto Co., St. Louis, Mo., and dibaurylphosphatidylcholinewas obtained from Serdary Research Laboratories, London,Ontario, Canada. Media for culturing the bacterial and marnmalian cells were obtained from Becton, Dickinson and Co.,Cockeysvibbe,Md., and Grand Island Biological Co., GrandIsland, N. Y., respectively. Fetal calf serum was obtained fromReheis Chemical Co., Inc., Kankakee, Ill. TPA was purchasedfrom Dr. Peter Borchert, University of Minnesota MedicalSchool, Minneapolis, Minn. Other commercially available biochemicals were obtained from Sigma Chemical Co., St. Louis,Mo.

PolycyclicHydrocarbonsand Their Derivatives.Bothpyrene and B(a)P were purchased from Aldrich Chemical Co.,Milwaukee, Wis. (<98% purity). 7,8-H2B(a)P (46) and B(a)PH4-9,i 0-epoxide (45 were prepared as previously described.dihydrobenzo(a)pyrene; B(e)P H4-9,i 0-epoxide, 9, 10-epoxy-9, 10, i 1,12-tetrahydrobenzo(e)pyrene; 9, i 0-H2B(e)P,9, i 0-dihydrobenzo(e)pyrene; 10, i i -H2-cycloheptapyrene, i 0, i i -dihydrocycloheptapyrene; 10, 1i -H2-cycloheptapyrene8,9-oxide, 10, i i -dihydrocycloheptapyrene 8,9-oxide; B(a)P-H4-9, 10-epoxide,9, i 0-epoxy-7,8,9, i 0-tetrahydrobenzo(a)pyrene; B(a)P 4 ,5-oxide, benzo(a)pyrene 4,5-oxide; B(a)P 7,8-diol-9, 10-epoxide, either or both diastereomerically related 7,8-dihydroxy-9, i 0-epoxy-7,8,9, i 0-tetrahydrobenzo(a)pyrenes derived from B(a)P 7,8-dihydrodiol; CPP 3,4-oxide, cyclopenta(c,d)pyrene 3,4-oxide; B(e)P9, i 0-diol-i 1,12-epoxide, benzo(e)pyrene 9, i 0-diol-i 1•i 2-epoxide.All compounds are racemic mixtures where enantiomers are possible.

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Biological Activity of CPP

igigigiioei amount of phosphatidylcholine and NADPH resulted in a highly

active monooxygenase system that had less than i % of theepoxide hydnase activity found in microsomes. No detectablemonooxygenase activity was found in the epoxide hydrase

78-H B[o]P preparatIon.Mutagenesis Assays with Bacteria. Strains TA98 and

TAi 00 of histidine-dependent S. typhimurium were obtainedI 0 0 fromDr.B.Ames,UniversityofCalifornia,Berkeley,Calif.,and

0 0 wereculturedasdescribed(26, 37). Intrinsicmutagenicitywasassessed by incubating specific compounds (added in i 5 jsbof

CYCLOPENTAIc,d]PYRENE dirnethyl sulfoxide) with 2 x i 08 bacteria suspended in 0.5 ml

of phosphate-buffered saline (5 mM potassium phosphate: iSOmM sodium chloride, final pH 7.0), for 5 mm at 37°beforeaddition of top agar. Mutagenesis experiments with microsomes as the source of the monooxygenase activity werebased on the procedure described by Ames et a!. (i ). NADP(2.0 @smol),glucose 6-phosphate (2.5 @smol),and glucose-6-phosphate dehydnogenase (i unit) were added to a i 3- x i 00-mm culture tube in 0.25 ml of pH 7.4 buffer consisting of SO@zmoIsodium phosphate, 4 @,tmolof MgCb2,and i 6.5 @rnoIofKCI. Microsomes were sterilized by filtration through a 0.45-@mfilter and were added in 0.25 ml of O.i 5 MKCI. The bacteria

I -PYRENYLOXIRANE (2 x i 08) were added in 0. i ml of phosphate-buffered saline.

Reactions were started by the addition of the compound in i S@Iof acetone:dimethyb sulfoxide (9:i ), and the complete mix

tunes were incubated at 37°for S mm before the addition oftop agar. Experiments with the purified monooxygenase systemwere conducted as previously described (42) with 300 units ofNADPH-cytochnome c reductase and 0.05 @smolof phosphatidylcholine per incubation. After incubation of the histidinedependent bacteria with onwithout either enzyme system, 2 mlof top agar were added, and the entire mixture was pouredonto a histidine-deficient agar Petnidish. Mutations to histidineindependent growth were assessed by counting the macroscopic colonies of bacteria after a 2-day incubation of theplates at 37°.All experiments were performed in triplicate.

Mutagenesis Assays with Mammalian Cells. Line V79-6 ofChinese hamster cells (3) was a generous gift of Dr. E. H. Y.Chu, University of Michigan, Ann Arbor, Mich. The cells, which

PYRENE B[oJP

@i@c1oi@iw @TgTgT9,I0―H2B[e]P Io,II-H2cYcL0HEPTAPYRENE

i6ei

I -VINYLPYRENE

ig@gx4 -VINYLPYRENE I - PYRENYLGLYCOL

TgTgT

0

b@OO

BIa}P H4-9,IO-EPOXIDE B[ej P H4-9,I0-EPOXIDE

?>fl@j 0CIôiôjw @mT@TgT4 -PYREPIYLOXIRANE 10,11-H2CYCLOHEPTAPYRENE8,9-OXIDE

Chart 1. Structures of the compounds used in this study. Absolute stereochemistry isnotimplied. According to the International Union ofPure and AppliedChemistry. 10,1 1-H@-cycloheptapyreneand i 0, 11-H@-cycIoheptapyrene8,9-oxide would be named 7,8-H2-cyclohepta[c,d)pyrene and 7,8-H2-cyclohepta[c,d]pyrene 9,10-oxide.

Synthesis of 9,10-H2B(e)P, B(e)P H4-9,i 0-epoxide, i 0, i i -H2-cycloheptapyrene, and i 0,i 1-H2-cycboheptapyrene 8,9-oxidewas as described (20). 1-Pynenyboxiranewas prepared frompyrene-1-carboxabdehyde as described (47) and was hydrolyzed with aqueous acid to i -pyrenylglycol. A related Wittigreaction of the carboxabdehyde with triphenyl methyl phosphonium bromide provided i -vinylpyrene. 4-Pyrenyboxinanewas prepared in a manneridenticalto that usedfor the synthesis of 9,1O-dimethyl-2-anthryboxinane(47) except that 4-acetylpyrene (9) was used as the starting material. n-Butyl lithiumand Fed3 (8) were used to effect the deoxygenation of 4-pyrenyboxirane to 4-vinylpyrene. All 5 of the above 1- and 4-substituted pyrene derivatives were judged pure from theirnuclear magnetic resonance and mass spectra. CPP was prepared as described (i 3).

Microsomal Enzymes. Hepatic rnicnosomeswere obtainedfrom immaturemale Long Evansrats pretneatedwith the polychlorinated biphenyl mixture Arocbor 1254. The cytochromesP-450 induced by Arocbon 1254 represent a mixture of themajor hemoproteins induced by phenobarbitab and 3-methylcholanthrene (29, 30). This cytochrome P-450 mixture waspurified from the microsomesas described (30). NADPH-cytochrome c reductase (48) and epoxide hydrase (24) werepurified from liven microsomes of immature male Long Evansrats pretreatedwith sodiumphenobarbitalas described in theindicated references. Reconstitution of NADPH-cytochrorne creductase and hemoprotein in the presence of the appropriate

appear to be devoid of the enzymes that metabolize polycyclichydrocarbons, were cultured as described previously (37).Four replicate culture dishes (60 mm) were each seeded withi 02 cells to evaluate toxicity, and i 6 replicate culture dishes(60 mm) were each seeded with i x i O@cells to assessinduction of 8-azaguanine-resistant colonies.

Tumorigenicity Studies. Female Charles RivenCD-i micewere obtained at 6 weeks of age from Charles River MouseFarms, North Wilmington, Mass., were housed in polycarbonate boxes with corncob bedding, and were fed a commercialdiet (Purina laboratory chow; Ralston Purina Co., St. Louis,Mo.) and water ad libitum. At 8 weeks of age, the mice wereanesthetized with ether and were shaved on the dorsal surfacewith electric clippers. Four days later, the mice received asingle topical application of the test compounds in 200 @lofsolvent. Control animals received only solvent. Each treatmentgroup consisted of 30 mice, housed i S animals per 9- x i 8-x 6-inch pobycarbonate box. The TPA tumor promoter (i 6

nmob/200 ,sl of acetone) was applied twice weekly to eachmouse, commencing 7 days after administration of the testcompounds. Incidence of papilbomaswas recorded at biweeklyintervals.

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375 75 5 30HYDROCARBON (nemi)

A. Wood eta!.

of the most active compound when microsomes were thesource of monooxygenase activity, and it had oven90% of theactivity of the most active compound when the purified monooxygenase system was utilized.

9, i 0-H2B(e)Pwas efficiently metabolized to mutagenic products under all experimental conditions, but particularly so withstrain TA98, where it was either the most active substrate onhad 98% of the activity of the most active substrate.

CPP was metabolized to products highly mutagenic to bothstrain TA98 and strain TAi 00 when microsomes were thesource of the monooxygenase system. Under these conditions,CPP had 77 to i 00% of the activity of the most active compound, and it was 4- to 7-fold more active than B(a)P. However,with the purified monooxygenase system, CPP had only 25and 8% of the activity of the most active hydrocarbon in strainsTA98 and TM 00, respectively. CPP was more efficiently activated to products rnutagenic to strain TA100 by the purifiedmonooxygenase system (23 His@'revertants/pmol cytochnomeP-4SO)than it was by micnosornes(i 0 His'@revertants/pmob ofcytochnome P-4SO).

The metabolic products of i 0, i i -H2-cycboheptapynenecon

8

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600

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644 CANCERRESEARCHVOL. 40

RESULTS

Metabolic Activation of Pyrene Derivatives to Mutagens.The ability of 7 hydrocarbons and i -pynenylglycol to be metabobically activated to mutagenic products was examined instrains TA98 and TA100 of S. typhimurium under conditionswhere (a) microsornal cytochnome P-4S0 was varied and thesubstrate concentration was kept constant (Chart 2), (b) microsomal cytochnome P-450 was kept constant and the concentration of hydrocarbon was varied (Chart 3), and (C)cytochromeP-4SOcontent of a highly purified and reconstituted monooxygenase system was varied and substrate concentration waskept constant (Chart 4). The dose-response relationships forthese 6 experiments are shown in the indicated charts, and therelative mutagenic activities of the metabolic products of the 8compounds are summarized in Table i.

The metabolic products of 7,8-H2B(a)Pwere more mutagenicto S. typhimurium TAi 00 than were the metabolic products ofany of the other hydrocarbons, regardless of the source of themonooxygenase system and the experimental conditions used.In strain TA98, 7,8-H2B(a)P had about one-half of the activity

Chart 2. Relationship between the metabolic activation of pyrene derivatives to products mutagenic to strains TA98 (A) and TA100(B) of S. typhimurium and the amount of microsomal cytochrome P-450 in the reactionmixture. The amount of substrate added was30 nmol. Background mutation frequencies of20 and 90 histidine revertants/plate for strainsTA98 and TA100, respectively, have beensubtracted from the data, which are presentedas the means of 3 replicate incubations.

Chart 3. Relationship between the microsomal activation of pyrene derivatives to products mutagenic to strains TA98 (A) and TA100(B) of S. typhimurium and the amount of substrate in the incubation mixture. Each incubation mixture contained 0. 10 nmol of microsomal cytochrome P-450. Background mutation frequencies of 32 and 135 histidine revertants/plate for strains TA98 and TA100,respectively, have been subtracted.

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Relative mutagenic activities of the metabolic products of the hydrocarbons in S.typhimuriumIneach experiment, mutagenic activities were calculated from the initial slopes of the dose-response curvesandexpressed

as either histidine revertants per nmol of cytochrome P-450 (Charts 2 and 4) or histidine revertants pernmolsubstrate(Chart 3). The most mutagenic compound in each experiment was assigned i 00% activity. The activity oftheother

hydrocarbons is expressed as a percentage of thisactivity.Microsomal

monooxygenase system Purified monooxygenase system varied en

Varied enzyme5 Varied substratebzymeCHydrocarbon

TA98 TA100 TA98 TA100 TA98 TM00B(a)P

20 20 13 NDd 8ND7,8-H2B(a)P44 iOO 51 100 941009,iO-H2B(e)P98 6i 100 69 10034CPPioo 77 85 80 2581

0, 11-H2-Cycloheptapyrene 27 i 5 45 26 9020i-Vinylpyrene35 15 31 26 50101

-Pyrenylglycol 0 1 0 0 104-Vinylpyrene2 5 3 4 5 3

Biological Activity of CPP

Chart 4. Relationship between the metebolic activation of pyrene derivatives to products mutagenic to strains TA98 (A) and TA100(B)ofS.typhimuriumandtheamountofhighlypurified cytochrome P.450 in the incubationmixture. Each assay contained 10 nmol ofsubstrate, and the incubation time was 5 mm.Background mutation frequencies of 16 and74 hlstidine revertants/plate in strains TA98and TA100. respectively, have been subtracted from the data.

p00

CYTOCHROME P450 (pmoi(

Table i

8Microsomal cytochrome P-450 was varied as indicated in Chart 2, and 15 nmol of hydrocarbon were added toeach incubation mixture.

b The amount of hydrocarbon was varied as indicated in Chart 3. and 0. 1 0 nmol of microsomal cytochrome P-450

was added to each incubation mixture.C Highly purified cytochrome P-450 was varied as indicated in Chart 4, and 10 nmol of hydrocarbon were added to

each incubation mixture.d ND, not determined.

sistently had higher mutagenicity toward strain TA98 thantoward strain TAI 00, and the hydrocarbon was very efficientlymetabolized to mutagenic products by the purified monooxygenase system. i -Vinylpynenewas metabolizedto moderatelyactive products which were more mutagenic to strain TA98than they were to strain TA100. In the former strain, i -vinylpyrene was more active than was B(a)P. i -Pyrenylglycolwasnot activated to products mutagenic to either strain of bacteria,and 4-vinylpynene only had from 6 to 30% of the activity of itspositional isomer, i -vinylpyrene.

Intrinsic Mutagenic Activity of the Polycyclic HydrocarbonEpoxidee. The intrinsic mutagenicity of i - and 4-pynenyboxiraneand I 0,1 i -H2-cycboheptapynene8,9-oxide instrainsTA98and TA100 of S. typhimurium is compared to the activity of thebay-regiontetnahydnoepoxidesof B(a)P and B(e)P in Chart S.Consistent with previous studies (42), B(e)P H4-9,i 0-epoxide5was more active than B(a)P H4-9,i 0-epoxide in strain TA98,whilethe conversewas true in strainTM 00. These resultsare

S B(e)P-H4-9,1 0-epoxide is formed by the oxidation of the 1 1 , 1 2-double bond

of 9,10-H2B(e)P. However, the epoxide is named so that the epoxide moiety isassigned the lowest numbers consistent with International Union of Pure andApplied Chemistry rules of nomenclature.

also consistent with the metabolic activation of 7,8-H2B(a)Pand 9,i 0-H2B(e)P, the metabolic precursors of these tetrahydroepoxides, described above. In strain TA98, i -pynenyboxirane was i S times more active than was 4-pyrenyboxinaneandhad activity comparable to that of B(a)P-H4-9,I 0-epoxide. Instrain TA100, i - and 4-pyrenyloxirane induced mutations atone-half and one-fourth the rate, respectively, observed instrain TA98; i -pyrenyboxirane was 27 times more active than4-pyrenyboxirane. The higher mutagenic activity of i -pyrenyloxirane in strain TA98 relative to strain TAi 00 is consistentwith the metabolic activation studies with i -vinylpyrene in these2 strains but differs from results obtained by Drinkwaten et a!.(5). i 0, i i -H2-cycboheptapynene8,9-oxide induced mutationsat the same rate as i -pyrenyboxirane in both strains TA98 andTA100. In Chinese hamster V79 cells, i 0,i i -H2-cycboheptapyrene 8,9-oxide was severalfold more active than was i -pyrenyboxirane, which induced mutations to 8-azaguanine resistance at a i 0-fold higher frequency than did 4-pyrenyboxirane (Chart 6). The higher intrinsic activity of i -pyrenyboxiranerelative to 4-pynenyboxiranein both strains of bacteria and themammalian cells is consistent with the metabolic activationstudies with i - and 4-vinylpynene described above.

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V@. @‘N 4-Pyrenyloxirone\‘@I-. 10,11-H2 Cycloheptapyrene 8,9-oxide

I I I I .1

lyzes an essential hydration leading to the formation of highlymutagenic and carcinogenic diol-epoxides, namely, the hydration of benzo-ring arene oxides to their corresponding dihydnodiols. While the diol-epoxides themselves are at best poorsubstrates for epoxide hydrase, the enzyme readily convertsK-region arene oxides and tetrahydnoepoxides of pobycyclichydrocarbons to nonmutagenic products (33, 37, 38, 42, 44).Chart 7 illustrates the effect of adding purified epoxide hydraseto the purified monooxygenase system prior to the metabolicactivation of B(a)P and CPP to products mutagenic to S.typhimurium strain TA100. The mutagenic activity of the metabobicproduct(s) of CPP was readily abolished by addition ofepoxide hydrase to the monooxygenase system. One unit ofepoxide hydrase diminished the mutation rate by oven 70%. Inmarked contrast to these results, addition of up to i 0 units ofepoxide hydrase had no effect on mutation frequency whenB(a)P was the substrate, suggesting that boss of mutagenicactivity resulting from hydration of B(a)P 4,5-oxide was babanced by the increased formation of B(a)P 7,8-diol-9,i 0-epoxides. Previous studies (4i , 42) which examined the effect ofpurified epoxide hydrase on the metabolic activation of B(a)Phave shown that the enzyme diminished the mutation rate by50% in strain TAi 538 and by 30% in strain TA98. The straindependent differences in the effect of epoxide hydnase areprobably due to the fact that the ratio of mutagenic activity ofthe B(a)P 7,8-diob-9,i 0-epoxides relative to B(a)P 4,5-oxide ishighest in strain TA100 (ratio, i 4), lowest in strain TA1538(ratio, 0.5), and intermediate in strain TA98 (ratio, 3).

The ability of highly purified epoxide hydnase to metabolizei - and 4-pyrenyboxinaneand i 0, i 1-H2-cycboheptapyrene 8,9-oxide to their corresponding nonmutagenic products is shownin Table 2. Since 1-pynenyboxiraneand cycboheptapyrene 8,9-oxide have the same mutation frequency in bacteria (Chart 5),they were tested at the same concentration. The data indicatethat 1-pyrenyboxirane is more efficiently converted to nonmutagenic products by epoxide hydnase than is i 0, 1i -H2-cycbohepatapynene 8,9-oxide. Although a relatively high dose of 4-

@ 100

5 10

A. Wood et a!.

IS 20

Di 02 03 04 05 01 02 03 04 05

EPOXIDE nmoi)

Chart 5. Intrinsic mutagenicity of the bay-region tetrahydroepoxides of B(a)Pand benzo(e)pyrene, i - and 4-pyrenyloxirane, and 10, 11-H2-cycloheptapyrene8,9-oxide (CHPO) in strains TA98 and TA100 of S. typhimurium. Indicatedamounts of the epoxides were incubated as described in ‘‘Materialsand Methods.‘‘Background mutation frequencies of 30 and 134 histidine revertants/platefor strains TA98 and TA100, respectively, have been subtracted from the data.Ordinate and abscissa le9ends for the insets are the same as those of the mainchart.

I00

80

60

40

20

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EPOXIDE(nmol/mI)Chart 6. Intrinsic cytotoxicity (top) and mutagenicity (bottom) of 10,1i -H2-

cycloheptapyrene 8,9-oxide, i -pyrenyloxirane, and 4-pyrenyloxirane in Chinesehamster V79 cells. Indicated amounts of the epoxides were incubated with thecells for 1 hr as described in “Materialsand Methods.―

Effect of Epoxide Hydrase on Mutagenic Activity of 5everal of the Hydrocarbons or Their Epoxides. The biologicalactivity expressed by a reactive metabobite is dependent, inpart, on its rate of formation relative to its spontaneous orenzyme-catalyzed rate of degradation. Epoxide hydnase cata

2 4 6 8 10EPOXIDE HYDRASE (units)

Chart 7. Effect of epoxide hydrase on the metabolic activation of B(a)P andCPPby a purified cytochrome P-450 monooxygenase system. The monooxygenase system was reconstituted as described in “Materialsand Methods,―and theindicated amounts of purified epoxide hydrase were added immediately after thecytochrome P-450 (75 pmol) and before the hydrocarbons (final concentration,25 saM).Strain TA100 of S. typhimurium was used, and the values are expressedas a percentage of the number of histidine revertants observed for each hydrocarbon in the absence of any added epoxide hydrase.

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Effect of epoxide hydrase on the mutagenicity of 1- and 4-pyrenyloxirane and10,11-H,-cycloheptapyrene8,9-oxidein strainTA98of S.typhimuriumMutagenic

activity@'at following doses of epoxide hy

drasecompound5

nmol 0 unitC 1 unit 5units1

-Pyrenyloxirane 0.3 735 59 4(iOO) (8)(0.5)4-Pyrenyloxlrane

2.0 325 i 07 25(100) (33)(8)10,1

1-H@-CycIoheptapyrene8,9- 0.3 769 337 48oxide (100) (44) (6)

Tumor-initiating activity of CPP, benzo(a)anthracene, and chrysene onmouseskinafter 26 weeks of tumor promotion withTPAThe

tumor study was conducted as described in “MaterialsandMethods―andthe legend to Table 3. According to the 4-fold contingency test ofMainlandandMurray (25), the percentage of mice with tumors must be equal to orgreaterthan27% and 33% for statistical significance at the 5% and 1% levels, respec

tively, when, as in the present case, the group size is 30 and a 4%tumorincidenceis observed in the solvent controlgroup.%

of mice with Tumors/mousetumors

1.02.5Compound@amol @cmoI 1.0 @zmol 2.5@&moISolvent

4 0.04 ±0.048CPP 30 37 0.47 ±0.1@ o.so ±o.iBenzo(a)anthracene 17 38 0.i7 ±0.07 0.67 ±Chrysene NDC 80 ND 2.16 ±0,3&'

Tumor-initiating activity of pyrene and pyrene derivatives on mouse skinafter26weeks of tumor promotion withTPAThirty

female CD-i mice received a single topical application of thecompoundsin2O0-@alof acetone. Seven days later, the mice started receivingtwice-weeklyapplications

of TPA (i 6 nmol/application) in 200 @alof acetone for 26weeks.From27 to 29 mIce in each group were alive at 26 weeks of promotion.Accordingto

the 4-fold contingency test described by Mainland and Murray (25),thepercentageof mice with tumors must be equal to or greater than 37% and43%for

statistical significance at the 5% and 1% levels, respectively, when, as inthepresentcase, the group size is 30, and a 10% tumor incidence is observed inthesolventcontrol group.%

of mice with tuTumors/mousemors0.1

0.4 0.i0.4Compound@amoI @zmol @zmol@amoISolvent

iO0.10Pyrene14 10 0.140.101

-Vinylpyrene 6 17 0.060.27i-Pyrenyloxirane18 24 0.180.411

-Pyrenylglycol 7 0 0.0704-Vinylpyrene7 21 0.070.2i4-Pyrenyloxirane0 7 00.07cPP

10 2i 0.100.2910.11-14.Cycloheptapyrene 11 23 0.1 10.23B(a)P

68 73 2.0 4.6

induced 4 times as many tumors per mouse as did the solvent,acetone. However, this activity was less than i 0% of the tumorinitiating activity of B(a)P. CPP followed i -pyrenyboxirane intumor-initiating activity. The time course for tumor induction byB(a)P and CPP is shown in Chart 8. The data (Table 3; Chart8) clearly show that, under the conditions of this experiment,the tumor-initiating activity of CPP is extremely weak relative tothat of B(a)P. When the background tumor frequency is subtracted, a dose of 0.4 jzmobof B(a)P is over 20 times moretumorigenic than is the same dose of CPP. A dose of 0.1 @mobof CPP induced 0.i tumor/animal, the same as the solvent,while B(a)P induced 2.0 tumors/animal. To better assess tumon-initiating activity, higher doses of CPP were comparedwith equivalent doses of the weak tumor initiatorsbenzo(a)anthracene and chrysene (Table 4). At both i .0- and2.S-@tmobdoses, CPPand benzo(a)anthracene have statisticallyinsignificant differences in tumor-initiating activity. In contrast,

-@— - .

2 8 24WEEKS OF PROMOTION

Chart 8. Time course of tumor incidence in female CD-i mice treated with asingle dose of B(a)P or CPP and subsequent twice-weekly applications of thetumor promoter TPA. Tumor incidence per mouse was calculated by dividing thetotal number of tumors observed per group by the total number of animals at riskin the group. No more than 2 animals in each group of 30 animals died during thecourse of the 28-week experiment. Solvent-treated control animals had anaverage of 0.1 tumor/animal, and this value has not been subtracted from thedata.

Table 4

5.0

4.0

3.0

2.0

//@ B[oJP,0.4ysmoI0

/ 8[o]P,o.I@mo)

CPP,0.4@mo)cpp,o.I @moI

Ui(I)

0

Ui0.

U)

0

I-a Indicated amounts of the epoxides selected from the linear regions of the

dose-response curves in Chart 5 were added in 15 @aIof dimethyl sulfoxide toapproximately 2 x 108 bacteria and the indicated amounts of highly purifiedepoxide hydrase. The bacteria and the enzyme were suspended and dissolved,respectively, in a total volume of 0.5 ml of 5 mM potassium phosphate:i 50 mMsodium chloride, final pH 7.0. After 5 mm at 37°,2 ml of top agar were added,and the cultures were plated as described in “Materialsand Methods.―

b Mutagenic activity of each compound in the absence and presence ofepoxide hydrase is expressed as the mean number of His@revertants observedper plate. Numbers in parentheses represent the percentage of mutagenic activityobserved for that compound in the absence of added epoxide hydrase. Allexperiments were performed in triplicate, and the background mutation frequencyof i 5 Hish revertants/plate has been subtracted.

C Units of epoxide hydrase are defined as nmol of styrene glycol formed from

styrene oxide per minute at pH 8.7.

Table 3

a Mean ±SE.b Significantly different from the solvent control value at p@ 0.05.

C ND, not determined. In a previous initiation-promotion study (2i ), 1 .25 @molof chrysene, applied in 200 galof tetrahydrofuran:dimethyl sulfoxide (95:5),induced a 43% tumor incidence with an average of 0.97 tumor/mouse.

d Significantly different from the value for CPP as well as the solvent control

value at p@ 0.05.

647

Biological Activity of CPP

pyrenyloxinane was used, the data in Table 2 indicate that themutagenic activity of this oxide is also readily abolished byepoxide hydrase.

Tumor-inItiating Activities of the Pyrene Derivatives onMouse Skin. The incidenceof skintumorsinfemaleCD-i miceafter a single topical application of 0.1 or 0.4 @smoIof thepyrene derivatives and 26 weeks of twice-weekly applicationsof the tumor promoter TPA is summarized in Table 3. With theexception of B(a)P, all the compounds were weak tumor initiators. Pyrene, 1-pyrenylglycol, and 4-pyrenyboxirane failed toshow a dose-response relationship or induce a tumor incidenceabove that observed in control animals treated with solvent andTPA. 1-Pyrenyboxirane was the most active of the pyrenederivatives [exclusive of B(a)P], and at a dose of 0.4 @zmolit

Table 2

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A. Wood eta!.

chrysene is clearly more active than both hydrocarbons in thistumor model.

DISCUSSIONThe bay-region theory of pobycyclic aromatic hydrocarbon

carcinogenicity proposes that benzo-ning diol-epoxides inwhich the epoxide group forms part of a bay region will be themost chemically and biologically active diol-epoxide for a givenhydrocarbon (14—i7). This high activity is rationalized by perturbationab molecular orbital calculations in terms of the easeof conversion of the diobepoxides to electrophibic carboniumions, with bay-region diob-epoxides being calculated to be themost reactive for a given hydrocarbon (i 6). Since the originalformulation of the theory, 8 hydrocarbons have been adequatebytested and have been found to comply with the predictions of the theory (cf. Refs. 4, 17, 22, and 40).

When we first described the molecular orbital calculationswith the diol-epoxides, we noted that non-bay-region diob-epoxides of certain hydrocarbons backing bay regions could havereactivity equal to or greater than bay-region diob-epoxidesderived from some carcinogenic hydrocarbons with bay regions (16). Nonetheless, and despite the additional fact thatCPP cannot form a benzo-ning diol-epoxide, CPP has beencited as an exception to the bay-region theory (6, i 8). CPP3,4-oxide, the putative metabobitesuggested to be responsiblefor the metabolism-induced mutagenicity of CPP (6), should behighly reactive. Ring opening can occur to yield a i -pyrenylcation, analogous to that derived from the highly reactive bayregion B(a)P-7,8-diob-9,i 0-epoxides, or a 4-pyrenyl cation,analogous to that derived from the B(e)P-9,i 0-diob-i i ,i 2-epoxides, with opening to the 1-pyrenyl cation expected to bemore favorable. Additionally, ring strain should enhance thereactivity of CPP 3,4-oxide and may contribute to the fact thatit has thus far eluded synthesis.6 In order to determine thestructural factors involved in the mutagenicity and carcinogenicity of CPP, a number of epoxides that can yield i - and 4-pyrenyl-substituted cations have been examined, along withtheir potential metabolic precursors and CPP (Chart i).

A summary of the metabolic activation of B(a)P, CPP, andthe related vinyl compounds is given in Table 1. Since 3 typesof metabolic activation were used with 2 tester strains, a simplestatement of the results is not possible. In agreement with theprevious study (6), CPP is a better substrate for formation ofmutagens, from 3- to 7-fold in the present study, than is B(a)P.In 5 ofthe 6 experiments, however, either 7,8-H2B(a)Por 9,i 0-H2B(e)Pwas the most active substrate in each group tested. Inthe sixth experiment, which varied the concentration of microsomal enzyme in the presence of S. typhimurium TA98, CPPand 9,i 0-H2B(e)P had the same activity. In the most extremecase, the purified monooxygenase system with strain TA100,CPP was i 2.5-fold less active than was 7,8-H2B(a)P. Thus, wemust conclude that there is nothing particularly unique aboutthe metabolic activation of CPP when it is compared to a seriesof rebated compounds. One additional interesting aspect ofTable 1 is that, when the vinyl group forms part of a fused ring,the compound is generally more active than the corresponding1- or 4-vinylpyrene.

Inherent mutagenicity of the various epoxides tested (Charts

6 During the preparation of this manuscript, Eisenstadt and Gold (7) reported

on the synthesis of CPP 3,4-oxide at the Fourth International Symposium onMicrosomes and Drug Oxidations in Ann Arbor, Mich. , July 15 to 18, i 979. Thecompound is highly mutagenic and very chemically reactive.

5 and 6) generally parallels the ability of their putative vinylprecursors to be activated to mutagens. The metabolicallyinduced mutagenicity of CPP is thought to be due to an epoxide, since very small amounts of epoxide hydrase (Chart 7)readily block this mutagenicity. Since pyrene K-region oxidehas less than 5% of the mutagenic activity of B(a)P 4,5-oxide(41), CPP 3,4-oxide does seem to be the most attractivestructure to account for the activity of CPP.6

On s.c. injection of 6.5 mg of CPP into 2S-g CFW/Jic mice,Neal and Tnieff (27) observed a 50% incidence of fibrosancomas at the site of injection after 244 days. Although crosscomparisons between different experiments cannot be madewith accuracy, it is clear that CPP is less active than is B(a)P,which produced an 80% tumor incidence after a single s.c.injection of 0.23 mg into 20-g C3H/fCUM mice (i 9). To testthe proposal of Eisenstadt and Gold (6) that CPP should behighly tumonigenic, based on its mutagenicity toward strains ofS. typhimurium, 9 structurally related compounds were cornpared in initiation-promotion experiments on mouse skin (Table3; Chart 8). At the high dose (0.4 @smob),B(a)P caused theformation of 4.6 papilbomas/mouse. The next most active cornpound was i-pyrenyloxirane, which caused 0.4i tumor/mouse. CPP produced only 0.29 tumor/mouse and was thusless than 5% as active as B(a)P. The very weak tumonigenicityof CPP may be due in part to the ability of epoxide hydrase toreadily detoxify the reactive metabolite (Chart 7), onthe metabolite may be intrinsically inactive as a carcinogen. The presentexperiments emphasize the hazards of attempting to predicttumor-initiating activity based on quantitative interpretation ofmutagenicity experiments with the S. typhimurium strains. Weare reminded of our previous studies of B(a)P 4,5-oxide andthe isomeric B(a)P 7,8-diol-9,i 0-epoxides. While all S of thecompounds were highly mutagenic to the S. typhimuriumstrains (36, 39). only one diob-epoxide enantiomen was tumorigenic in the newborn mouse and on mouse skin (2, 23, 3i,32,35).

ADDENDUMRecently, Newbold, Brookes and Harvey (Int. J. Cancer 24:

203—209,1979) reported that 1-pyrenyloxirane has greatercytotoxic activity than BP 7,8-diob-9,i 0-epoxide-2 (anti isomer)and mutagenic activity comparable to BP 7,8-diol-9,i 0-epoxide-i (syn isomer) in Chinese hamster V79 cells. These resultsare at variance with the data presented in this manuscript andpreviously reported (ref. 44 and Thakkeretal., Mob.Phanmacol.14: 5O2—5i3, i 978). The basis for the discrepancy may bethat Newbold et a!. used an exceedingly long exposure time(24 hr) to compare the biological activity of compounds withvery different stabilities in aqueous solutions. Thus, B[a]P 7,8-diol-9,i 0-epoxides-i and -2 have half-lives in tissue culturemedium of 0.5 and 9 mm, respectively (44) while the half-life of1-pyrenyboxiranein tissueculture mediumis 150 mm.

ACKNOWLEDGMENTSWe thank Ann Marie Williams for her assistance in the preparation of this

manuscript.

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649MARCH 1980

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c,dMutagenicity and Tumor-initiating Activity of Cyclopenta(

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