(CANCER RESEARCH 41, 2589-2597, July 1981]0008-5472/81 /0041 -OOOOS02.00

Mutagenicity of the Bay-Region Diol-Epoxides and Other Benzo-Ring

Derivatives of Dibenzo(a,/i)pyrene and Dibenzo(a,/)pyrene

Alexander W. Wood,1 Richard L. Chang, Wayne Levin, Dene E. Ryan, Paul E. Thomas, Roland E. Lehr2,Subodh Kumar, Dennis J. Sardella,3 Eliahu Boger, Haruhiko Yagi, Jane M. Sayer, Donald M. Jerina, and

A. H. Conney

Department of Biochemistry and Drug Metabolism, Hoffmann-La Roche Inc.. Nutley. New Jersey 07110 ¡A.W. W., R. L. C., W. L., D. E. R., P. E. T., A. H. C.¡;Department of Chemistry. University of Oklahoma. Norman. Oklahoma 73019 ¡R.E. L., S. K..]; Laboratory of Bioorganic Chemistry, National Institute of Arthritis.Metabolism and Digestive Diseases. NIH, Bethesda, Maryland 20205 [H. Y., J M. S.. D. M. J.]: and Department of Chemistry. Boston College. Chestnut Hill,Massachusetts 02167[D. J. S., E. B.J

ABSTRACT

The mutagenic activities of dibenzo(a,n)pyrene, diben-zo(a,/)pyrene, and a total of 11 of their benzo-ring derivatives

were evaluated in bacterial and mammalian cells in the absenceor presence of a mammalian metabolic activation system, frans-1,2-Dihydroxy-1,2-dihydrodibenzo(a,rt)pyrene and irans-3,4-dihydroxy-3,4-dihydrodibenzo(a,/)pyrene, the expected dihy-drodiol precursors of bay-region diol-epoxides, were metabolized by a cytochrome P-450-dependent monooxygenase sys

tem to products which were more mutagenic to strains TA98and TA100 of Salmonella typhimurium than were the metabolicproducts formed from their respective parent hydrocarbons.For each dihydrodiol, replacement of the benzo-ring double

bond adjacent to the diol moiety with a single bond resulted intetrahydrodiol derivatives which could not be metabolicallyactivated, suggesting that one or both diastereomeric bay-region diol-epoxides were the bioactivated metabolites. Theauthentic bay-region diol-epoxide diastereomers of diben-

zo(a,Mpyrene and dibenzo(a,/)pyrene in which the benzylichydroxyl group and the epoxide oxygen are frans (diol-epoxide

2 series) were highly mutagenic in strains TA98 and TA100 ofS. typhimurium and in cultured Chinese hamster V79 cells.Neither diol-epoxide was significantly, if at all, metabolized byepoxide hydrolase. The bay-region diol-epoxide of dibenzo(a,/)

pyrene was from 1.5 to 5 times more active as a mutagen thanthe diol-epoxide of dibenzo(a,rt)pyrene, and in strain TA98 of

S. typhimurium as well as Chinese hamster V79 cells, it hadactivity comparable to that of the highly carcinogenic bay-region diol-epoxide of benzo(a)pyrene.

INTRODUCTION

Polycyclic aromatic hydrocarbons are widespread environmental pollutants which, like many other chemical carcinogens,must be metabolized to their biologically active products. Extensive studies with the pentacyclic aromatic hydrocarbon,B(a)P," have revealed the metabolic pathway for the activation

Received January 8, 1981; accepted March 24, 1981.1To whom requests for reprints should be addressed.2 Supported in part by Grant CA 22985 from The National Cancer Institute.3 Supported in part by Grant CA 23454.4 The abbreviations used are: B(a)P, benzo(a)pyrene; DB(a,h)P, diben-

zo(a,f>)pyrene; DB(a,i)P, dibenzo(a,0pyrene; DMSO, dimethyl sulfoxide; 2,10-DFDB(a,i)P, 2,10-difluorodibenzo(a,/)pyrene; 3.4-H2 DB(a,h)P, 3,4-dihydrodi-benzo(a,h)pyrene; 1,2-H2 DB(a,i)P, 1,2-dihydrodibenzo(a,/)pyrene; DB(a,h)P 1 2-dihydrodiol, (rans-1,2-dihydroxy-1,2-dihydrodibenzo(a,ri)pyrene; DB(a,h)P 1,2-diol-3,4-epoxide 2, (±)-1/?,2a-dihydroxy-3a,4a-epoxy-1,2,3,4-tetrahydrodi-benzo(a.rt)pyrene; DB(a,i)P 3.4-dihydrodiol. (rans-3,4-dihydroxy-3.4-dihydrodi-benzo(a./)pyrene; DB(a.i)P 3,4-diol-1,2-epoxide 2, (±)-3a,4/J-dihydroxy-1a,2a-

of this hydrocarbon and have shown that a bay-region diol-epoxide,5 ( + )-7/S,8a-dihydroxy-9a,1 Oa-epoxy-7,8,9,10-tetra-

hydrobenzo(a)pyrene, is the principal ultimate carcinogenicmetabolite (3, 25). Subsequent studies with almost a dozenother pentacyclic and tetracyclic aromatic hydrocarbons haverevealed that formation of bay-region diol-epoxides on angularbenzo-rings is a common pathway by which both unsubstitutedand alkyl-substituted hydrocarbons exert their mutagenic andtumorigenic effects (6, 13, 22, 34).

Several considerations suggested that an evaluation of thebiological activity of the hexacyclic aromatic hydrocarbonswould be of interest. DB(a,h)P and DB(a,i)P (Chart 1) are bothpotent carcinogens (2, 4, 7, 8, 10, 29) which have been foundin significant concentrations in polluted environments (9, 15,20). Additionally, quantum mechanical calculations designedto predict the ease of triol carbonium ion formation from diol-epoxides indicated that the bay-region diol-epoxides of

DB(a,h)P and DB(a,i)P should have particularly high chemicalreactivity (12). The present study evaluates the mutagenicityof a bay-region diol-epoxide diastereomer of both hydrocarbons, their dihydrodiol precursors, and several other benzo-

ring derivatives of the dibenzopyrenes. The structures of thecompounds studied are shown in Chart 1.

MATERIALS AND METHODS

Materials. Aroclor 1254 (lot KG 12-638) was obtained fromMonsanto Co., St. Louis, Mo., and dilauroyl phosphatidylcho-

line was obtained from Serdary Research Laboratories, Inc.,London, Ontario, Canada. Media for culturing the bacterial andmammalian cells were obtained from Becton Dickinson andCo., Cockeysville, Md., and Grand Island Biological Co., GrandIsland, N. Y., respectively. Fetal calf serum was obtained fromReheis Chemical Co., Kankakee, III. Other commercially available biochemicals were obtained from Sigma Chemical Co., St.Louis, Mo. DMSO was distilled from calcium hydride underreduced pressure and stored under an argon atmosphere inamber bottles.

epoxy-1,2.3,4-tetrahydrodibenzo(a.0prene; DB(a,i)P H«-3,4-diol, frans-3.4-dihy-droxy-1.2.3,4-tetrahydrodibenzo(a,/)pyrene; DB(a,h)P H4-1,2-diol, trans-i ,2-di-hydroxy-1,2,3.4-tetrahydrodibenzo(a.ft)pyrene; DB(a,i)P H«-3,4-epoxide, 3,4-epoxy-1,2,3,4-tetrahydrodibenzo(a.0pyrene; DB(a,h)P H4-1,2-epoxide, 1,2-epoxy-1,2,3,4-tetrahydrodibenzo(a.Wpyrene. All compounds are racemic mixtures where enantiomers are possible.

5 The hydroxyl groups of dihydrodiols produced by mammalian microsomalepoxide hydrase have trans relative stereochemistry. The diol-epoxides derivedfrom the dihydrodiols exist as diastereomeric pairs in which the benzylic hydroxylgroup and the epoxide oxygen are either c/s (isomer 1 series) or trans (isomer 2series). Since each diastereomer can be resolved into 2 enantiomers, 4 stereo-isomerie diol-epoxides are possible.

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A. W. Wood et al.

3.4-H2 DB[o.n]P DBto.nlP M4-I.2-EPOXIDE

Sûûù:DBto.nJP D8lo,n]P 1,2-OlMTDRODlOL DBto.hlP I.2-DIOL-3.4-

EPOXIDE-2

DB[0,1] P M4- 1,2-DlOL

l.2-H2 OBIo.O P DBto.i]Pn4-3,4-EPOXIDE

OB[o,i]P 3.4-DIMYDROOIOL DB10..JP 3.4-OIOL-I.2-EPOXIDE- 2

2.10-OF OB[o.,]P DB[o.i)P H4-3.4-DIOL

Chart 1. Structures of DB(a,h)P, DB(a,i)P. and their derivatives studied in thisinvestigation. Both parent hydrocarbons are symmetrical molecules, and the 2bay-regions of each compound are identical. Diol-epoxides of polycyclic hydrocarbons formed by mammals exist as diastereomeric pairs in which the epoxidemoiety and the benzylic hydroxyl are either cis (diol-epoxide 1 series) or trans(diol-epoxide 2 series). Only the trans diastereomers which are shown here werestudied. Stereochemistry is relative.

Polycyclic Hydrocarbons and Their Derivatives. DB(a,i)Pwas purchased from ICN Pharmaceuticals, Inc., Plainview,N. Y. The sample was applied to a silica gel column and elutedwith benzene:hexane (1:1) to remove small amounts of impurities. DB(a,h)P was obtained from Chemical Procurement Laboratories, Inc., College Point, N. Y. The hydrocarbon waschromatographed as above and crystallized from xylene (m.p.308°). 2,10-DFDB(a,i)P was synthesized as described previ

ously (2). Purification consisted of chromatography on a Whatman Magnum-9 ODS-2 column eluted with acetonitrile at 10

ml/min. The hydrocarbon emerged at 2.8 column volumes.Due to its low solubility, only 2 mg of compound in 0.2 ml oftetrahydrofuran could be injected on each pass. Five recycleswere required for each injection to remove a closely relatedimpurity. All 3 hydrocarbons were judged to be essentially purebased on Chromatographie, mass spectral, and nuclear mag

netic resonance analysis. 3,4-H2 DB(a,h)P, 1,2-H2 DB(a,i)P,DB(a,h)P 1,2-dihydrodiol, DB(a,h)P 1,2-diol-3,4-epoxide 2,DB(a,i)P 3,4-dihydrodiol, and DB(a,i)P 3,4-diol-1,2-epoxide 2

were synthesized as described previously (16).Synthesis of DB(a,i)P H4-3,4-diol and DB(a,h)P H4-1,2-diol.

To 40 mg of frans-3,4-diacetoxy-1,2,3,4-tetrahydrodiben-

zo(a,0pyrene (16) were added 5 ml tetrahydrofuran, 5 mlmethyl alcohol, and 2.5 ml 1 N NaOH. The mixture was stirredat 25° for 3 hr under N2. Most of the solvent was removed

under reduced pressure, and the solid residue was washedwith water and hot benzene and filtered to give 20 mg of theproduct, m.p. 233-235°. DB(a,h)P H4-1,2-diol, m.p. 255-257°, was prepared in a similar manner.

Synthesis of DB(a,i)P H4-3,4-epoxide and DB(a,h)P H4-1,2-epoxide. To a stirred solution of 1,2-H2 DB(a,i)P (16) (100 mg)in tetrahydrofuran (50 ml), under N2, were added 500 mg of m-chloroperoxybenzoic acid (85%) at 25°. After 2.5 hr, the

mixture was diluted with ether and then was extracted with ice-cold 10% KOH, followed by cold water. The ether phase wasdried over MgSO4, filtered, and evaporated to give a light-yellow solid that was triturated with cold ether to give a light-yellow solid (80 mg) of m.p. 181-183°. In a like manner,

DB(a,h)P H4-1,2-epoxide was obtained in 80% yield, m.p. 205-207°.All compounds gave the expected microanalysis, nuclear

magnetic resonance spectra, and/or molecular ions in theirmass spectra.

Microsomal Enzymes. Hepatic microsomes were obtainedfrom immature male Long Evans rats treated with the polychlo-rinated biphenyl mixture Aroclor 1254. The cytochromes P-450 induced by Aroclor 1254 represent a mixture of the majorhemoproteins induced by phénobarbital and 3-methylcholan-threne (23, 24). This cytochrome P-450 mixture was purifiedfrom the microsomes as described (24). NADPH-cytochrome

c reducíase(37) and epoxide hydrolase (19) were purified fromliver microsomes of immature male Long Evans rats treatedwith sodium phénobarbital as described. Reconstitution of theNADPH-cytochrome c reducíaseand hemoprotein in the pres

ence of the appropriate amount of phosphatidylcholine andNADPH resulted in a highly active monooxygenase system thathad less than 1% of the epoxide hydrolase activity found inmicrosomes. No cytochrome c reducíaseor cytochrome P-450was detecled in purified epoxide hydrolase.

Mutagenesis Assays with Bacteria. Slrains TA98 andTA100 of hislidine-dependenl Salmonella typhimurium (21)

were oblained from Dr. B. Ames, Universily of California, Berkeley, Calif., and were cullured as described (31). Intrinsic mu-

tagenicity was assessed by incubating Ine epoxides (added in15 fi\ of DMSO) with 2 x 10s bacteria suspended in 0.5 ml of

phosphale-buffered saline (5 mw potassium phosphate: 150rriM sodium chloride, final pH 7.0) for 5 min at 37° before

addition of top agar. Mutagenesis experimenls with microsomes as the source of monooxygenase activity were basedon the procedure described by Ames ef al. (1). NADP (2.0/imol), glucose 6-phosphale (2.5 /¿mol),and glucose-6-phos-phale dehydrogenase (1 unii) were added to a 13- x 100-mm

culture tube in 0.25 ml of pH 7.4 buffer consisting of 50 /imolsodium phosphate, 4 /¿molof MgCI2, and 16.5 /¿molof KCI. Thebacleria (2 x 108 cells) were added in 0.1 ml of Ihe phosphate-

buffered saline. Reactions were started by addilion of Ihe lesicompound in 15 /tl of acetone:DMSO:NH4OH (6:4:0.01), andIhe complete mixtures were incubated at 37°for 5 min before

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the addition of top agar. Experiments with the purified mono-

oxygenase system were conducted as described previously(35) with 300 units of NADPH-cytochrome c reductase and

0.05 fimo\ phosphatidylcholine in each reaction mixture. Aftera 10-min incubation of the histidine-dependent bacteria withthe enzyme-activating system, 2 ml of top agar was added, andthe entire mixture was poured onto a histidine-deficient agarRetri dish. Mutations to histidine-independent growth were

assessed by counting the macroscopic colonies of bacteriaafter a 2-day incubation of the plates at 37°. All experiments

were performed in triplicate.Mutagenesis Assays with Mammalian Cells. Line V79-6 of

Chinese hamster cells (5) was a generous gift of Dr. E. H. Y.Chu, University of Michigan, Ann Arbor, Mich. The cells, whichappear to be devoid of the enzymes that metabolize polycyclichydrocarbons, were cultured as described previously (31).Assays assessing the cytotoxicity and intrinsic mutagenicity ofthe diol-epoxides were adapted from the procedure of Chu (5)

and were performed as described previously (31). The V79cells were seeded in 60-mm culture dishes at concentrationsof 1 x 102 (4 dishes) and 1 x 104 (16 dishes) cells/dish for

experiments assessing cell survival and mutagenicity, respectively. After an 18-hr culture in 5 ml of medium, the testcompound was added in 20 /¿Iof anhydrous DMSO. After a 1-hr incubation at 37°, the medium and test compound were

removed from the culture dish, and the cells were washed oncewith 5 ml of phosphate-buffered saline (9.5 mw sodium phos

phate: 140 HIM sodium chloride:3 rriM potassium chloride, final

Mutagenicity of Dibenzopyrene Derivatives

pH 7.2) and then cultured in 5 ml of fresh medium. To assesscell survival, culture dishes which contained 102 cells wereincubated at 37° for 7 days, at which time the macroscopic

colonies of cells were fixed with methanol, stained with Giemsa,and counted. To assess mutagenic activity, the culture dishescontaining 10" cells were incubated at 37°for 3 days. At this

time, mutant cells resistant to 8-azaguanine were selected byaddition of 10 /ig of 8-azaguanine per ml of culture medium.

On Day 7, the medium was replaced with 5 ml of fresh mediumcontaining the same amount of 8-azaguanine. One week later,colonies resistant to 8-azaguanine were fixed with methanol,

stained with Giemsa, and counted.Measurement of Hydrolysis Rates. Kinetics of the hydrolysis

of diol-epoxides of DB(a,h)P and DB(a,i)P were followed at 25°

in 10% dioxane in water [ionic strength, 0.10 M (NaCIO4)]either by spectrophotometric measurements or by high-pres

sure liquid Chromatographie analyses of aliquots of the reactionmixture following trapping of unreacted diol-epoxide as its

mercaptoethanol addition product. Wavelengths used for thespectrophotometric kinetic studies were 303 and 390 nm forthe diol-epoxides of DB(a,h)P and DB(a,i)P, respectively.

RESULTS

Metabolic Activation of Dibenzopyrene Derivatives to Bacterial Mutagens. The mutagenic activities of the metabolicproducts formed by incubation of DB(a,h)P, DB(a,i)P, and 7 oftheir derivatives with a cytochrome P-450-dependent mono-

oxygenase system are shown in Charts 2 to 4 for both strains

400-

KLU

300 -

200 -

IOO -

025 050 075CYTOCHROME P-450 (nmol)

0.6CYTOCHROME P-450 (nmol)

Chart 2. Dependence of the metabolic activation of DB(a,h)P, DB(a,i)P, and their derivatives to products mutagenic to strains TA98 W and TA100 (B) of Styphimurium on the amount of hepatic microsomes in the incubation mixture. The amount of substrate in the 0.6-ml incubation mixture was 12.5 nmol. Backgroundmutation frequencies of 25 and 107 His' revertants/plate for strains TA98 and TA100. respectively, have been subtracted from the data which are presented as the

means of 3 replicate determinations. •¿�,DB(a,h)P; O, DB(a,i)P; A. DB(a,h)P 1,2-dihydrodiol; A, DB(a.i)P 3,4-dihydrodiol; V, DB(a,h)P H,-1,2-diol; V, DB(a,i)P H.-3.4-diol; •¿�3,4-H2 DB<a.h)P; D, 1,2-H2 DB(a,i)P; X, 2,10-DFDB(a,i)P.

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A. W. Wood et al.

TA98 (A) and TA100 (S) of S. typhimurium. In Chart 2, 12.5nmol of the compounds were incubated with varying amountsof hepatic microsomes from Aroclor 1254-treated rats. In bothstrains of bacteria, the metabolic products of DB(a,h)P 1,2-dihydrodiol were 4- to 10-fold more active than those of the

parent hydrocarbon and were the most active of any of themetabolites formed from the other DB(a,h)P derivatives. Removal of the 3,4 double bond in DB(a,h)P 1,2-dihydrodiolyielded a compound, DB(a,h) H4-1,2-diol, which was not metabolized to mutagenic products. 3,4-H2 DB(a,h)P was meta-

bolically activated to a significant extent in strain TA98 but notin strain TA100. DB(a,i)P 3,4-dihydrodiol induced the highest

level of mutations of any of the compounds tested although itsuniquely high activity relative to all the other DB(a,i)P derivatives was seen only in strain TA100. In strain TA98, the metabolic products formed from DB(a,i)P and 2,10-DFDB(a,i)P in

duced mutations at a slightly greater initial rate than didDB(a,i)P 3,4-dihydrodiol. As was found with the analogoustetrahydrodiol of DB(a,h)P, DB(a,i)P H4-3,4-diol could not be

metabolically activated in either strain of bacteria.Chart 3 summarizes the results of experiments in strains

TA98 and TA100 of S. typhimurium in which the concentrationof hepatic microsomes was held constant at 0.5 nmol of cyto-chrome P-450 and the hydrocarbons and their derivatives werevaried over more than a 10-fold range in concentration. The

optimal amount of substrate was between 6 and 12 nmol in the0.6-ml incubation mixture. With the exception of 2,10-

DFDB(a,i)P in strain TA98, amounts of substrate higher than

12.5 nmol did not result in further activation of the mutageniccompounds and did not result in activation of the otherwiseinactive compounds. DB(a,i)P 3,4-dihydrodiol was clearly the

most active compound in strain TA98 as well as strain TA100.Otherwise, the same pattern of relative activity of the compounds was observed when substrate concentration was varied(Chart 3) as was just described when microsomes were variedand substrate concentration was held constant (Chart 2). Thedownturn in mutagenic activity observed with both strains ofbacteria at the 12.5-nmol dose of DB(a,i)P 3,4-dihydrodiol is

most likely due to production of toxic amounts of the mutagenicmetabolite, although no macroscopic or microscopic (X100)irregularities in the background lawn of bacteria were observed.

Chart 4 summarizes the results of experiments in both strainsof bacteria when a highly purified and reconstituted hepaticmonooxygenase system was utilized to metabolically activatevarying amounts of the 9 dibenzopyrene derivatives. Comparison of the amounts of substrate and cytochrome P-450 utilized

to achieve the mutation frequencies shown in Charts 3 and 4clearly indicates that the purified monooxygenase system wasa more efficient activating system than washed microsomes(per nmol cytochrome P-450) for the dihydrodiols and several

of the other active compounds. Comparison of the mutationfrequencies obtained with the 2 sources of the monooxygenasesystem (Charts 3 and 4) reveal, with one exception, a similarpattern of activation of the compounds. 2,10-DFDB(a,i)P which

had relatively high activity in strain TA98 when microsomes

400 -

orlu

300 -

200

100

6125 125 250DIBENZPYRENE DERIVATIVE (nmol)

50.0

800-

o:ui

o:+v>I

600-

400-

200-

6.125 125 250

DIBENZPYRENE DERIVATIVE (nmol)

500

Chart 3. Effect of substrate concentration on the metabolic activation of DB(a,h)P, DB(a,i)P, and their derivatives catalyzed by hepatic microsomes from Aroclor1254-treated rats. Each incubation mixture contained 0.5 nmol of cytochrome P-450. Background mutation frequencies for strains TA98 (A) and TA100 (B) of S.typhimurium were 24 and 88, respectively, and have been subtracted from the data. See legend to Chart 2 for the key to symbols.

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Mutagenicity of Dibenzopyrene Derivatives

2000 -

1500 -

o.\(/)

UJa:

1000-

500

2.5 5.0 7.5 10

DIBENZPYRENE DERIVATIVE ( nmol )

2.5 5.0 7.5 10

DIBENZPYRENE DERIVATIVE (nmol)

Chart 4. Metabolic activation of DB(a,h)P, DB(a,i)P, and their derivatives by a highly purified and reconstituted monooxygenase system. The amount of cytochromeP-450 in each incubation was 0.075 nmol, the substrate concentration was as indicated on the abscissa, and the amounts of the other components of themonooxygenase system are described in "Materials and Methods." Incubation time was 10 min. Background mutation frequencies for strains TA98 (A) and TA 100

(ß)of S. typhimurium were 40 and 101, respectively, and have been subtracted from the data. See legend to Chart 2 for the key to the symbols.

were utilized as the source of the monooxygenase system(Chart 3A) was relatively less active in the presence of thepurified monooxygenase system (Chart 4A). The dihydrodiolderivatives of both DB(a,h)P and DB(a,i)P were always moreactive in strain TA100 than in strain TA98, and this wasparticularly evident when the purified monooxygenase systemwas utilized for activation of these compounds.

The relative mutagenic activity of all 9 compounds whichwere tested in the presence of a metabolic activation system issummarized in Table 1. Calculations were made both frominitial rates of mutation induction as well as from the maximalmutation frequency observed (numbers in parentheses), andall data are expressed as a percentage of the highest activityobserved in each test system.

Intrinsic Mutagenicity of the Dibenzopyrene Epoxide Derivatives. Dose response curves of the intrinsic mutagenicactivity of the bay-region diol-epoxide 2 diastereomers of

DB(a,h)P and DB(a,i)P in strains TA98 and TA100 of S. typhimurium are illustrated in Chart 5. In strain TA98, DB(a,h)P 1,2-diol-3,4-epoxide 2 induced mutations at a rate of 580 His*

revertants/nmol while DB(a,i)P 3,4-diol-1,2-epoxide 2 inducedmutations at a 3-fold higher rate of 1940 revertants/nmol. Bothdiol-epoxides produced higher mutation frequencies in strainTA100 compared to strain TA98. The bay-region diol-epoxideof DB(a,h)P induced a mutation frequency of 4900 His* re

vertants/nmol while the bay-region diol-epoxide of DB(a,i)Pinduced mutations at a rate of 7700 His* revertants/plate in

strain TA100. These results are consistent with the metabolicactivation studies reported above in that the dihydrodiol ofDB(a,i)P was activated to mutagenic products to a greater

extent than the dihydrodiol of DB(a,h)P in experiments utilizingeither strain of bacteria and that the metabolic products of bothdihydrodiols produced higher mutation frequencies in strainTA100 relative to strain TA98. Chart 5 also illustrates that thenon-bay-region tetrahydroepoxides of both DB(a,h)P andDB(a,i)P are very weak mutagens in both strains of bacteria.

The intrinsic cytotoxic and mutagenic activity of the diben-zopyrene bay-region diol-epoxides in Chinese hamster V79

cells is illustrated in Chart 6. A 1.2 /IM concentration ofDB(a,h)P 1,2-diol-3,4-epoxide 2 and a 0.45 fiM concentrationof DB(a,i)P 3,4-diol-1,2-epoxide 2 killed 50% of the culturedmammalian cells. Both diol-epoxides induced mutations to 8-azaguanine resistance in a linear dose-dependent manner. Onan equal concentration basis, the bay-region diol-epoxide ofDB(a,i)P was 5- to 6-times more active than the bay-regiondiol-epoxide of DB(a,h)P. At equitoxic doses, the difference inactivities was about 2-fold with the diol-epoxide of DB(a,i)Pagain being more active. The non-bay-region tetrahydroepox

ides of DB(a,h)P and DB(a,i)P had minimal activity in the V79cells and induced less than 20 8-azaguanine-resistant colonies/105 surviving cells at concentrations up to 30 nmol/ml

(data not shown).Biological Half-Life and Chemical Reactivity of Diol-Epox-

ides. The mutagenic activity of the dibenzopyrene diol-epox

ides decreased monoexponentially as a function of incubationtime at 37°in complete tissue culture medium (pH 7.2 to 7.6;

0.5% DMSO). The half-lives of the diol-epoxides are summarized in Table 2 and can be compared to an average half-life of9 min for the bay-region diol-epoxide 2 isomer of B(a)P (36).The data in Table 2 also show that the dibenzopyrene bay-

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A. W. Wood et al.

Table 1

Summary of the relative mutagenic activity of 9 dibenzopyrene compounds tested in strains TA98 and TA100 of S.typhimurium in the presence of a metabolic activation system

Values were calculated from the initial slopes of the dose-response curves and the highest absolute mutationfrequencies observed, either peak or plateau values (numbers in parentheses). Data are expressed as a percentage ofthe most active DB(a,h)P and DB(a.i)P derivative.

Metabolic activation system

Varied microsomal P-450,fixed substrate3

Varied substrate, fixedmicrosomal P-450fi

Varied substrate, fixedpurified P-450C

Compound TA98 TA100 TA98 TA100 TA98 TA100

DB(a,h)P3.4-HjDB(a,h)PDB(a,h)P

1,2-dihydrodiolDB(a,h)PH4-1.2-diolDB(a,i)P1

,2-H2DB(a,i)PDB(a,i)P3,4-dihydrodiolDB(a,i)PrV3,4-diol2.10-DFDB(a,i)P"

Data from Chart2.6Data from Chart3.0Data from Chart4."Tho wt.M.tw f\i riOtn i\I18(8)81

(72)100(100)14(11)98

(78)43(40)84(100)5(2)1

00 (50)14(8)4(8)100(100)15(8)36(31)16(10)100(100)15(6)15(10),.

. , .25

(20)100(76)100(100)13(13)20(100")10(41)100(93)2(7)10(74)15(14)11(10)100(100)11

(10)17(19)5(5)100(100)5(5)7(11),

. .56(51)75

(69)100(100)6(9)51

(57)40(49)100(100)11

(11)23(22)17(13)10(13)100(100)9(8)14(13)13(13)100(100)6(5)5(5)no/o

;\D T ¿_

dihydrodiol (see Chart 3/0.

400 -

- 300 -UJI-

è»200 -

ce•¿�f

lOO-

OB[o.h ] Pl,2-DIOL-3,4-EPOXIOE-2

2000 -

OO

1500 -

1000-

500-

03 0.3 0.4

EPOXIDE ( nmol)

1.0

DB[O,I]P3,4-DIOL-l,2-EPOXIDE-2

DB [o,h] P,2-DIOL-3,4-EPOXIDE-2

02 0.3 0.4EPOXIDE (nmol)

Chart 5. Intrinsic mutagenic activity of the bay-region diol-epoxide 2 diastereomers and the non-bay-region tetrahydroepoxides of DB(a.h)P and DB(a,i)P in strainsTA98 (Ai ana TA 100 (B) of S. typhimurium. Approximately 2X10" bacteria of either strain were treated with the indicated amounts of the epoxides as described in"Materials and Methods." Each value represents the average number of histidine revertan! colonies observed per plate in 3 replicate determinations. Spontaneousmutation frequencies of 47 and 90 His' revenants/plate for strains TA98 and TA100. respectively, have been subtracted from the data.

region diol-epoxides are resistant to hydrolysis catalyzed byepoxide hydrolase. Similar results with epoxide hydrolase havebeen obtained for all other polycyclic hydrocarbon diol-epoxides thus far tested (26, 31 -33, 35, 36). The mutagenic activityof benzo(a)pyrene 4,5-oxide, a K-region arene oxide, wasreadily abolished by epoxide hydrolase, indicating that theenzyme was catalytically active under the conditions of theexperiment.

Kinetic constants for the hydrolysis of the diol-epoxides of

DB(a,h)P, and DB(a,i)P, and B(a)P in 10% dioxane in water(ionic strength, 0.1 M) follow the rate law

where aH»represents antilog (-pH), and are given in Table 3.This same rate law is followed for the acid-catalyzed (kH»)andspontaneous (k0) hydrolysis of several other bay-region diol-

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Mutagenicity of Dibenzopyrene Derivatives

epoxides (28). Comparison of these above 3 compounds showsa clear trend of increasing activity towards spontaneous hydrolysis (k0) for the series B(a)P, DB(a,h)P, and DB(a,i)P.

100

Table 3Kinetic constants for the hydrolysis of bay-region diol-epoxides

Rates were determined at 25°in 10% dioxane in water in 0.1 M NaCIO«.Rate

constants were determined from pH rate profiles.

D8[o,i]P 3,4-DIOL-l,2-EPOXIDE -2

OB[o,h]P l,2-DIOL-3,4-EPOXIDE-2

0.5 I.O I.5

DIOL EPOXIDE (nmol/ml)

Chart 6. Intrinsic cytotoxicity (fop) and mutagenicity (bottom) of the bay-region diol-epoxide 2 diastereomers and the non-bay-region tetrahydroepoxidesof DB(a,h)P and OB(a.i)P in Chinese hamster V79 cells. Cells were treated withthe indicated amounts of the diol-epoxides for 1 hr at 37° as described in"Materials and Methods." The spontaneous mutation frequency of solvent-treated cultures was 0 to 1.3 8-azaguanine-resistant colonies/105 surviving cells,

and the plating efficiency of the cells was between 82 and 90%.

Table 2Half-lives of the bay-region diol-epoxides of DB(a,h)P and DB(a,i)P in tissue

culture medium and the effect of epoxide hydrolase on their mutagenic activity

% of control with the following epoxide hydrolase

units0

Compound3DB(a,h)P

1,2-diol-3,4-epoxide 2DB(a,i)P 3,4-diol-1 ,2-epoxide 2Benzo(a)pyrene, 4,5-oxidefl/2(min)2112.50100

100100192

9820598109 2

a Amounts of the epoxides were selected from linear portions of their dose-

dependent mutagenic response curves with strain TA100 of S. typhimurium." Epoxides were preincubated in Eagles' minimal essential medium containing

10% fetal calf serum, pH 7.2 to 7.6, and 37°.After various periods of time (0.5

to 120 min), 0.1-ml aliquots were tested for mutagenic activity toward strainTA100 of S. typhimurium as described in "Materials and Methods." The muta

genic activity of the epoxides decreased in a monoexponential fashion as afunction of preincubation time, and half-lives were calculated from these plots.

' Intrinsic mutagenicity of the epoxides in the presence of highly purifiedepoxide hydrolase was determined by the same procedure described in "Materials and Methods" for assessing intrinsic mutagenicity of the compounds towards

the bacteria. Epoxide hydrolase (1 unit is equal to 1 nmol of styrène glycolformed from styrène oxide per min at pH 8.7) was added to the bacteria beforethe epoxides.

Diol-epoxide(isomer2)DB(a.i)P

DB(a.h)PB(a)p'>Acid-catalyzed

hydro-lylsisa(kH» M"1sec'')2000

±100850 ±150

1400 ±200Spontaneous

hydrolysis8(k0sec"1)12.0

±3.0 X 10™43.2 ±0.3 x KT*1.3 ±0.5 X 10"*

Variations given for the rate constants represent the range in values forindividual determinations.

6 Data are taken from Ref. 27.

DISCUSSION

The bay-region diol-epoxide 2 diastereomer of both DB(a,h)P

and DB(a,i)P had high intrinsic mutagenic activity in bacterialand mammalian cells. The dihydrodiol precursors of these diol-

epoxides were metabolically activated to bacterial mutagens toa greater extent than their parent hydrocarbons, while theirtetrahydrodiol analogs, which cannot form bay-region diol-epoxides, were inactive. These results indicate that the bay-

region theory of polycyclic hydrocarbon carcinogenicity canbe extended to the hexacyclic aromatic hydrocarbons.

Perturbational molecular orbital calculations (AEdeioc/ß)havebeen used to predict the chemical reactivity of diol-epoxides

and tetrahydroepoxides of polycyclic hydrocarbons (12). Highvalues of AEdei0c/ßare indicative of the ability of a hydrocarbonto disperse the positive charge of the carbonium ion formedfrom the epoxide throughout the ^-electron system of the

hydrocarbon and predict high chemical reactivity. The valuesof AEdeioc/ßfor the bay-region diol-epoxides of DB(a,h)P and

DB(a,i)P, 0.845 and 0.866, respectively, are the highest calculated values for any hydrocarbon whose diol-epoxides have

been studied to date. By way of comparison, the value ofAEaeioc/ßfor the highly mutagenic and carcinogenic bay-regiondiol-epoxide 2 isomer of B(a)P is 0.794. The chemical reactivityof these diol-epoxides, as measured by their rate constants

(k0) for neutral hydrolysis in 10% dioxane in water (Table 3), isin good agreement with predictions based on AEdei0c//3. Thediol-epoxide of DB(a,i)P is approximately 4 times more reactivethan the diol-epoxide of DB(a,h)P and about 10 times morereactive than the diol-epoxide of B(a)P. At pH 7.2 in thedioxane:water system, the diol-epoxides of DB(a,i)P, DB(a,h)P,and B(a)P have half-lives of 9, 31, and 53 min, respectively.

Under these conditions, the predominant pathway of reactionis via uncatalyzed hydrolysis although acid-catalyzed hydroly

sis makes a significant contribution (36%) to the rate for thediol-epoxide of B(a)P. The biological half-lives of 12.5 and 21min for the diol-epoxides of DB(a,i)P and DB(a,h)P, respec

tively, are in keeping with the quantum mechanical calculationsand the values obtained in aqueous dioxane. However, theB(a)P diol-epoxide has a much shorter half-life (9 min) in tissue

culture medium than predicted. The complexity of the tissueculture medium which contains 10% fetal calf serum and numerous potential nucleophiles has been pointed out previously(36). With respect to DB(a,h)P and DB(a,i)P, the quantummechanical calculations are predictive of relative mutagenicactivity. The bay-region diol-epoxide of DB(a,i)P was 3.3-,1.6-, and 4.8-fold more active than its DB(a,h)P counterpart in

strains TA98 and TA100 and in V79 cells, respectively. Inaddition, the dihydrodiol precursor of the bay-region diol-epoxide of DB(a,i)P was always more active, in the presence of a

JULY 1981 2595

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A. W. Wood et al.

monooxygenase system, than its DB(a,h)P counterpart. Theintrinsic mutagenicity of the bay-region diol-epoxide 2 isomer

of B(a)P was determined in the same experiments (data notshown) which analyzed the activity of the dibenzopyrenes(Charts 5 and 6). While DB(a,i)P 3,4-diol-1,2-epoxide 2 hadessentially the same activity as the diol-epoxide 2 isomer of

B(a)P in strain TA98 of S. typhimuhum and in Chinese hamsterV79 cells and essentially the same half-life in tissue culturemedium, it has only 40% of the activity of the diol-epoxide 2

isomer of B(a)P in strain TA100 of S. typhimurium. It is notclear what factor(s) is responsible for the lower than predictedactivity of the dibenzopyrene diol-epoxides relative to the diol-

epoxide 2 isomer of B(a)P. Relative and absolute stereochemistry, as well as conformational factors, have been shown tomarkedly effect mutagenic and carcinogenic activity of hydrocarbon epoxides (13, 14, 17, 30, 33), and these factors arenot considered by the quantum mechanical calculations. Therelatively large size and low solubility of the dibenzopyrenemolecules may also be attenuating their biological activity. Itshould be kept in mind that the bay-region diol-epoxide (isomer

2) of B(a)P is one of the most potent mutagens yet described(11, 18, 36); thus, bay-region diol-epoxides of DB(a,h)P and

DB(a,i)P are very potent mutagens.Quantum mechanical calculations have also predicted that

non-bay-region diol-epoxides and tetrahydroepoxides shouldhave substantially less chemical reactivity than their bay-regioncounterparts (12). Although the non-bay-region diol-epoxides

of DB(a,h)P and DB(a,i)P were not available for the presentstudy, the very low mutagenic activity of DB(a,h)P H4-1,2-epoxide and DB(a,i)P H4-3,4-epoxide are certainly consistent

with the quantum mechanical calculations. It is of interest that3,4-H2 DB(a,h)P, the expected metabolic precursor of the non-bay-region tetrahydroepoxide, was metabolically activated to

a significant extent to products mutagenic to strain TA98 of S.typhimurium. 2,10-DFDB(a,i)P was also metabolically activated

by the microsomal enzyme system to products which hadrelatively good mutagenic activity in strain TA98 and relativelypoor activity in strain TA100. The basis for these interstraindifferences in S. typhimurium is not known. However, 2,10-

DFDB(a,i)P has little or no tumorigenic activity when injectedS.C. in C57BL/6J mice (2). All of the compounds studied inthis investigation are presently being evaluated for tumorigenicactivity, and the results of these studies will determine whichmutagenesis test system is the best predictor of biologicalactivity in vivo.

ACKNOWLEDGMENT

We thank Ann Marie Williams for her assistance in the preparation of thismanuscript.

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1981;41:2589-2597. Cancer Res   Alexander W. Wood, Richard L. Chang, Wayne Levin, et al.   )pyrene

a,i)pyrene and Dibenzo(a,hBenzo-Ring Derivatives of Dibenzo(Mutagenicity of the Bay-Region Diol-Epoxides and Other

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