[CANCER RESEARCH 45, 6406-6442, December 1985]

Comparative Metabolic Activation in Mouse Skin of the Weak Carcinogen6-Methylchrysene and the Strong Carcinogen 5-Methylchrysene1

Shantu Amin,2 Keith Huie, Assieh A. Melikian, Joanna M. Leszczynska, and Stephen S. Hecht

Way/or Dana Institute for Oisease Prevention, American Health Foundation, Valhalla, New York 10595

ABSTRACT

We compared the metabolic activation in mouse skin of theweak carcinogen 6-methylchrysene, which lacks a bay regionmethyl group, and the strong carcinogen 5-methylchrysene,which has a bay region methyl group. Metabolites of 6-methyl

chrysene were prepared using liver homogenates and wereidentified by their spectral properties and by comparison tosynthetic standards as dihydrodiols, hydroxymethyl derivatives,and phenols; their relative levels of formation in liver homogenates from rats and mice were dependent on inducer pretreatment. In mouse skin in vivo, the major metabolite of 6-methylchrysene was frans-1,2-dihydro-1,2-dihydroxy-6-methylchry-sene (6-MeC-1,2-diol), the precursor to a bay region dihydrodiolepoxide. Its concentration was greater than that of trans-1,2-dihydro-1,2-dihydroxy-5-methylchrysene (5-MeC-1,2-diol)formed in mouse skin from 5-methylchrysene. Since 5-MeC-1,2-diol has been identified as a major proximate carcinogen of 5-methylchrysene, the further metabolism and tumorigenicity of 5-MeC-1,2-diol and 6-MeC-1,2-diol were compared. Both dihydrodiols were converted to 1,2,3,4-tetraols and to 1,2-dihydroxymetabolites to similar extents in mouse skin. However, 5-MeC-1,2-diol was significantly more active than was 6-MeC-1,2-diol

as a tumor initiator on mouse skin. The formation of DMA adductsin mouse skin from 5-methylchrysene and 6-methylchrysene was

compared. Both hydrocarbons gave qualitatively similar adducipatterns, but the formation of dihydrodiol epoxide type adductswas 1/2oas great from 6-methylchrysene as from 5-methylchry

sene. The results of this study indicate that the weak tumorigenicity of 6-methylchrysene compared to that of 5-methylchry

sene is not due to differing rates of formation or further metabolism of their 1,2-dihydrodiols but is a likely consequence of thelower activity of 1,2-dihydroxy-3,4-epoxy-1,2,3,4-tetrahydro-6-methylchrysene compared to 1,2-dihydroxy-3,4-epoxy-1,2,3,4-tetrahydro-5-methylchrysene; the unique structural feature of thelatter is the presence of a methyl group and an epoxide ring inthe same bay region.

INTRODUCTION

The position of methyl substitution in methylated PAH3 pro-

Received 5/21/85; revised 9/3/85; accepted 9/4/85.1This study was supported by National Cancer Institute Grant CA 32242. This

is paper 89 of the series "A Study of Chemical Carcinogenesis."

2To whom requests for reprints should be addressed.3The abbreviations used are: PAH, polynuclear aromatic hydrocarbons; 6-MeC,

6-methylchrysene; 5-MeC, 5-methylchrysene; HPLC, high-performance liquid chro-

matography; MS, mass spectrum; NMR, nuclear magnetic resonance; s, singlet;d, doublet; t, triplet; m, multiplet; (vT, molecular ion; 6-MeC-1,2-diol, frans-1,2-dihydro-1,2-dihydroxy-6-methylchrysene; 5-MeC-1,2-diol, frans-1,2-dihydrc-1,2-dihydroxy-5-methylchrysene; 6-MeC-1,2,3,4-tetraols, isomers of 1,2,3,4-tetrahy-drc-1,2,3,4-tetrahydroxy-6-methylchrysene; 6-MeC-1,2-diol-3,4-epoxide, 1,2-dihy-droxy-3,4-epoxy-1,2,3,4-tetrahydro-6-methylchrysene; 5-MeC-1,2-dic4-3,4-epox-ide, 1,2-dihydroxy-3,4-epoxy-1,2,3,4-tetrahydro-5-methylchrysene.

foundly influences tumor-initiating and carcinogenic activity. 6-

MeC (Chart 1) is a weak tumor initiator and carcinogen on mouseskin, with activity comparable to that of chrysene, whereas 5-

MeC is a strong tumor initiator and carcinogen, with activitycomparable to that of benzo[a]pyrene (1, 2). The other fourmonomethylchrysenes are also only weakly active (1). Examplesof particularly tumorigenic monomethyl isomers are known inmany PAH systems (3, 4). Among alternant PAH, the structuralrequirements favoring tumorigenic activity are a bay regionmethyl group and a free peri position, both adjacent to anunsubstituted angular ring (4, 5). The enhancing effect on PAHtumorigenicity of a bay region methyl group adjacent to anunsubstituted angular ring has been observed in many systems,including phenanthrene, benz[a]anthracene, chrysene, 15,16-dihydro-11 -methylcyclopentafajphenanthren-l 7-one, benzo[a]-pyrene, cholanthrene, dibenz|a,fr]anthracene, and dibenz[a,y']an-

thracene (4-12). In order to explore the mechanistic basis for

the enhancing effect on tumorigenicity of a bay region methylgroup, we have compared the metabolic activation of 5-MeC,which has a bay region methyl group, and 6-MeC, which lacks

this structural feature.

MATERIALS AND METHODS

Apparatus. HPLC was carried out with a Waters Associates, Inc.,Model ALC/GPC-204 high-speed liquid Chromatograph equipped with a

Model 6000A solvent delivery system, a Model 660 solvent programmer,a Model U6K septumless injector, and a Model LC-25 UV/visible detector.Systems 1-3 were used for analysis. In System 1, a 4.0 x 250 mmLJchrosorb RP-18 10-^m column (EM Reagents, Cincinnati, OH) wasprogrammed as follows: a linear 20-min gradient from 30-50% methandin H2O, followed by a 1-h linear gradient from 50-100% methanol in H2O(flow rate, 3 ml/min). In System 2, two 4.6 x 250 mm LJchrosorb Si-6010-/i(Ti columns (EM Reagents) were eluted with hexane:tetrahydrofuran

(75:25) at a flow rate of 3 ml/min. In System 3, 2 Whatman Partisil PXS10/25 ODS columns (Whatman, Inc., Clifton, NJ) were eluted with aprogram as follows: a linear 30-min gradient from 35% methanol in H2Oto 55% methanol in H2O, then a 20-min isocratic period using 55%methanol in H2O, then a 20-min linear gradient to 100% methanol (flow

rate, 1.5 ml/min). UV spectra were run in methanol on a Cary Model 118spectrometer. MS were obtained with a Hewlett-Packard Model 5982A

instrument. Proton NMR spectra were obtained with a Jeol Model FX90Qspectrometer or a Brucker Model WM 250 instrument, courtesy ofProfessor Koji Nakanishi, Columbia University, or a Nicolet MagneticsNTC 300 Wide Bore Spectrometer, at Rockefeller University.

Chemicals. 6-MeC was prepared as described previously (1). 6-MeC-1,2-diol was isolated from incubation mixtures of 6-MeC and 9000 x gsupernatant from livers of Aroclor-pretreated rats as described below.6-MeC-1,2-diol was characterized by NMR and MS (see text). 6-MeC-1,2,3,4-tetraols were prepared by oxidation of 6-MeC-1,2-diol (0.001mmol) with m-chloroperbenzoic acid (0.026 mmol) in dry tetrahydrofuran

at room temperature for 2 h. The resulting dihydrodiol epoxide wasisolated, redissolved in tetrahydrofuran, and hydrolyzed in 10 ml ofcacodylate buffer, pH 7, for 12 h at room temperature. The 6-MeC-

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METABOLIC ACTIVATION OF 6-MeC AND 5-MeC

6-MeC S-MeCChart 1. Structures of 6-MeC and 5-MeC.

1,2,3,4-tetraols were extracted with ethyl acetate and purified by HPLC

(System 1). The retention times of the major and minor isomers were 20and 25 min, respectively. The MS of each isomer had a molecular ion atm/e 310. 5-MeC-1,2-diol was synthesized (13) and kindly provided by

Dr. Ronald G. Harvey, University of Chicago.[3H]6-MeC-1,2-diol and ["HJS-MeC-l ,2-diol were prepared metaboli-

cally using 9000 x g supernatant from livers of Aroclor-pretreated rats;5-MeC-1,2-diol formed in this way has been characterized previously(14). These dihydrodiols were >99% pure by HPLC. [3H]6-MeC was

prepared by catalytic tritium exchange (New England Nuclear, Boston,MA) of 12-bromo-6-MeC, obtained as follows. A suspension of 6-MeC

(48 mg) in acetic acid (5 ml) was heated under reflux for 10 min. Asolution of bromine (160 mg) in 2 ml of acetic acid was then addeddropwise, and refluxing was continued for 30 min. The product separatedas a crystalline material, mp 223-224°C (40 mg). NMR spectrum: ¿2.9

(s, 3H), 7.6-7.9 (m, 4H), 8.1-8.25 (m, 1H), 8.35-8.49 (m, 1H), 8.5 (s,1H), 8.6-8.85 (m, 2H), and 9.0 (s, 1H); MS m/e (relative intensity): 322(M*, 92.5), 320 (M+, 100), and 239 (86.3). The crude [3H]6-MeC was

purified by preparative thin-layer chromatography on a 20 x 20 cm silica

gel plate, using a solvent system of hexane:CH2Cl2 (80:20), Rf = 0.59.This yielded [3H]6-MeC with purity greater than 99%, 37.2 Ci/mmol. The

UV spectrum, HPLC retention times and R( value were identical to thoseof unlabeled 6-MeC.

6-Hydroxymethylchrysene was prepared as illustrated in Chart 2A. A

solution of phenylacetic acid (2.7 g, 0.02 md; Aldrich Chemical Co.,Milwaukee, Wl), 1-naphthaldehyde (3.1 g, 0.02 mol; Aldrich), 5 ml of

Methylamine, and 5 ml of acetic anhydride was heated with stirring at150°Cfor 6 h. After cooling, the dark solution was diluted with HaO (150

ml) and acidified with 50 ml concentrated HCI. The resulting suspensionwas extracted with CHCI3 (2 x 150 ml). The CHCI3 layer was washedwith H2O, dried (MgSO4), and concentrated to afford 3.0 g of 2-phenyl-3-(1-naphthyl)propenoic acid, m.p. 149-151 °C.MS m/e (relative intensity): 274 (M+, 50.5) and 229 (100).

A mixture of the above acid (2.7 g, 0.01 mol), K2CO3 (2.7 g, 0.02 mol),and dimethyl sulfate (1.8 g, 0.015 mol) in 150 ml acetone was heatedunder reflux for 4 h. The reaction mixture was then filtered, and theK2CO3 was washed several times with CHzCfe. The organic layer waswashed with H2O, dried (MgSO4), and evaporated to give the crudeester. This was purified by chromatography on silica gel with a solventsystem of hexane-CH2CI2 (80:20) to give 2.5 g (89%) of pure methyl-2-phenyl-3-(1-naphthyl)propenoate, m.p. 127-128°C. NMR spectrum:

53.85 (s, 3H), 6.9-7.3 (m, 7H), 7.4-7.9 (m, 4H), 8.05-8.2 (m, 1H), and8.45 (s, 1H); MS m/e (relative intensity): 288 (M+, 56.4) and 229 (100).

A solution of this ester (1.0 g, 0.034 mol) and iodine (5 mg) in drybenzene was stirred, and dry air was bubbled through the solution. Thiswas irradiated for 6 h at room temperature with a Hanovia 450-Wmedium-pressure mercury lamp, using a Pyrex filter. Removal of the

solvent gave 800 mg of a light yellow compound which was recrystallizedfrom ethanol to give pure 6-carbomethoxychrysene (0.5 g, 51%), m.p.147-148°C. NMR spectrum: «4.1(s, 3H), 7.6-7.8 (m, 5H), 7.9-8.1 (m,

2H), 8.65-8.9 (m, 3H), and 8.95-9.15 (m, 1H); MS m/e (relative intensity):286 (M+, 100), 255 (51 ), 227 (40), and 226 (53).

To a slurry of lithium aluminum hydride (36 mg, 0.001 mol) in ether(100 ml) at 0°Cwas added 6-carbomethoxychrysene (0.28 g, 0.001 mol)

in ether (10 ml). The reaction mixture was allowed to stir at 0°Cfor 1 h.

Ethyl acetate was added slowly, and the reaction mixture was acidified

with 1 N HCI. The organic phase was separated, collected, washed withsaturated aqueous NaCI, dried (MgSO4), and concentrated to yield 6-hydroxymethylchrysene as a white solid (0.2 g, 77%). It was recrystallized from methanol, m.p. 207°C. NMR spectrum: ¿5.0(t, 1H, OH), 5.25

(d, 2H, CH2), 7.4-7.8 (m, 4H), 7.95-8.1 (m, 2H), 8.13-8.3 (m, 1H), and8.6-8.9 (m, 4H); and MS m/e (relative intensity): 258 (M+, 100) and 229

(100).

C19H,40

Calculated: C 88.34, H 5.46Found: C 88.11, H 5.39

7-Hydroxy-6-MeC was prepared by a method similar to that illustratedin Chart 2A for 6-hydroxymethylchrysene. The starting materials were o-methoxyphenylacetic acid and 1-naphthaldehyde. The esterified condensation product was photocyclized to give 6-carbomethoxy-7-methoxy-chrysene. Reduction with UAIH4 gave 6-hydroxymethyl-7-methoxychry-

sene. This was oxidized with pyridinium chtorochromate and then reduced with hydrazine and KOH to give 7-methoxy-6-MeC. 7-Hydroxy-6-MeC was obtained by treating 7-methoxy-6-MeC with BBr3 in CHzCt.

NMR spectrum: 53.18 (s, 3H), 6.85 (d, 1H, H8, J8.9= 7.3 Hz), 7.47 (t, 1H,H9, J8,9= 7.3 Hz, J9.10= 7.2 Hz), 7.55-7.74 (m, 2H), 7.85-7.95 (m, 2H),8.45- 8.55 (s+d, 2H), 8.55 (d, 1H), and 8.72 (d, 1H); and MS m/e (relativeintensity): 258 (M+, 100).

The method used for the synthesis of 1-hydroxy-6-MeC is summarizedin Chart 2B. A solution of 1-bromomethyl-4-methylnaphthalene(15) (2.34

g, 0.01 mol) and 2.6 g (0.01 mol) of triphenylphosphine (Aldrich ChemicalCo.) in 50 ml of benzene was heated under reflux for 5 h. The reactionmixture was cooled, and the phosphonium salt (1.5 g) was isolated byfiltration.

To a stirred solution of this salt (0.9 g, 0.002 mol) and o-anisaldehyde(0.27 g, 0.002 mol; Aldrich Chemical Co.) in 50 ml of methanol wasadded sodium methoxkte (0.1 g, 0.002 mol). The mixture was stirred atroom temperature for 1 h and then diluted with H2O and extracted withCH2CI2. The organic solution was washed with H2O, dried (MgSO4), andconcentrated. The resulting oil was purified by silica gel chromatographywith a solvent system of hexane-CHjCb (80/20). This procedure gavecis- and irans-1-(4-methyl-1-naphthyl)-2-(2-methoxyphenyl)ethylene, as

an oil; NMR spectrum: 52.6 (s, 0.9H, cis- or frans-CH3), 2.65 (s, 2.1 H,cis- or frans-CH3), 3.75 (s, 0.9H, c/s- or frans-OCH3), 3.85 (s, 2.1 H, c/s-or frans-OCH3), 6.8-7.8 (m, 10H), 7.9-8.05 (m, 1H), and 8.15-8.3 (m,1H); and MS m/e (relative intensity): 274 (M+, 100), 259 (63 2) and 243

(39.3).The photochemical conversion of this alkene (100 mg, 0.0036 mol) to

1-methoxy-6-MeC was accomplished in 40% yield by the procedure

ICH,0)Z SO, __

«2«>3

CMjCOj» C02H

COjCHj C02CHJ CH2OH

Chart 2. Synthesis of (A) 6-hydroxymethylchrysene and (B) 1-hydroxy-6-MeC.

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METABOLIC ACTIVATION OF 6-MeC AND 5-MeC

described above for preparation of 6-carbomethoxychrysene. The crude

product was purified by column chromatography on silica gel with asolvent system of CH2CI2/hexane. 1-Methoxy-6-MeC (38 mg) was isolated as a crystalline solid, m.p. 159-160°C. NMR spectrum: 52.8 (s,

3H), 3.95 (s, 3H), 6.9 (d, 1H), 7.3-7.8 (m, 3H), 8.0-8.15 (m, 1H), and8.25-8.5 (m, 5H); and MS m/e (relative intensity), 272 (M+, 100) and 257

(35.4).

C20H160

Calculated: C 88.20, H 5.92Found: C 87.94, H 6.18

A solution of 1-methoxy-6-MeC (13 mg, 0.05 mmol) in 5 ml of CH2CI2was stirred under N2, and boron tribromide (1 M solution in CH2CI2, 0.5ml, Aldrich Chemical Co.) was added dropwise at room temperature.The reaction mixture was stirred for 1 h, diluted with H2O, and extractedwith CH2CI2. The organic solution was washed with H2O, dried (MgSO4),and concentrated to afford 1-hydroxy-6-MeC, which was pure according

to HPLC analysis. NMR spectrum: 63.1 (s, 3H, CH3), 6.9 (d, 1H, H2, J2.3= 7 Hz), 7.5 (t, 1H, H3, J2.3= J3.4= 7 Hz), 7.65-7.8 (m, 2H), 8.15 (d, 1H),

8.35-8.45 (m, 2H), 8.55 (s, 1H, H5), 8.61 (d, 1H), and 8.85 (d, 1H); andMS m/e (relative intensity): 258 (M+, 100).

1,2-Dimethoxy-6-MeC was prepared by the same sequence describedfor 1-methoxy-6-MeC, except that the starting material was 2,3-dime-

thoxybenzaldehyde. The NMR spectrum of the intermediate alkene wasas follows: 2.65 (s, 1H, c/s- or frans-CH3), 2.7 (s, 2H, c/s- or frans-CH3),3.72 and 3.85 (s, 2H, c/s- or frans-CH3), 3.79 and 3.81 (s, 4H, c/s- or

trans-OCH3), and 6.5-8.3 (m, 11H). The photochemical conversion ofthe alkene (75 mg, 0.25 mmol) to 1,2-dimethoxy-6-MeC was carried out

as described above. The crude compound was purified by columnchromatography on silica gel with a solvent system of CH2CI2-hexane(30/70) to afford 1,2-dimethoxy-6-MeC as a solid (19 mg, 25%), m.p.201-202°C. NMR spectrum: ¿2.85(s, 3H), 4.1 (s, 6H), 7.42 (d, 1H), 7.6-

7.8 (m, 2H), and 8.1-8.4 (m, 6H); and MS m/e (relative intensity): 302

(MM 00) and 287 (23.6).

Calculated: C 83.41 , H 5.99Found: C 83.20, H 6.13

Treatment of 1,2-dimethoxy-6-MeC with BBr3 in CH2CI2 as describedabove gave 1,2-dihydroxy-6-MeC. NMR spectrum: 52.8 (s, 3H), 6.5 (d,1H), and 6.7-8.8 (m, 8H); and MS, m/e (relative intensity): 274 (M+, 33.6),

244 (100), and 215 (44.8). An aliquot of 1,2-dihydroxy-6-MeC was

converted to the corresponding diacetate by treatment with acetic anhydride (0.5 ml) and pyridine (0.1 ml) at room temperature for 3 h. MSm/e (relative intensity): 358 (M+, 63.4) and 316 (100).

NADP+ and glucose 6-phosphate were obtained from Sigma Chemical

Co., St. Louis, MO. Aroclor 1254 was procured from Analabs, Inc.,Hamden, CT. Phénobarbital (sodium salt) was obtained from Mallinck-

rodt, St. Louis, MO. 3-Methylcholanthrene was purchased from EastmanKodak Co., Rochester, NY. 12-O-Tetradecanoylphorbol-1 3-acetate was

obtained from Consolidated Midland Corp., Brewster, NY.Animal Treatments for in Vitro Metabolism. Male F344 rats (190 to

230 g) and female CD-1 mice (20 to 30 g) were obtained from ChartesRiver Breeding Laboratories, Portage, Ml. Rats were treated 5 days priorto sacrifice with an i.p. injection of Aroclor 1254 in corn oil, 500 mg/kg.Mice were treated 48 h prior to sacrifice with an i.p. injection of 3-

methylcholanthrene in com oil (75 mg/kg). Control mice received injections of corn oil 5 days or 48 h prior to sacrifice, respectively.

Metabolism in Vitro. Metabolites of 6-MeC were produced in quan

tities sufficient for spectral identification by carrying out incubations asfollows. A mixture of 20 mg of 6-MeC in 4.0 ml dimethyl sulfoxide, 3.2ml of 0.4 M MgCI2, 3.2 ml of 1.65 M KCI, 0.8 ml of 1.0 M D-glucose 6-phosphate, and 6.4 ml of 0.1 M NADP+ was brought to a total volume of

80 ml with potassium phosphate buffer (pH 7.4), and 80 ml of 9000 x gsupernatant from the livers of Aroclor-pretreated rats were added. The

resulting mixture was added to two 500-ml Ertenmeyer flasks andincubated with shaking at 37°for 20 min, after which each flask was

treated with 80 ml acetone. The combined mixtures were extracted 4times with 160 ml ethyl acetate. The ethyl acetate layers were combined,dried (MgSO<), and concentrated at reduced pressure to a residue whichwas dissolved in methanol. Metabolites were analyzed by HPLC, usingSystems 1 and 2.

Metabolism studies of [3H]6-MeC (1.86 mg, 0.0077 mmol, 1 mCi/

mmol) were carried out by incubating the hydrocarbon with cofactorsand 9000 x g supernatant from mouse liver, exactly as described above,except on one-tenth the scale. Protein concentrations were 34.2 mg/mlfor 3-methylcholanthrene-induced mice and 38.8 mg/ml for control mice.

Metabolites were separated by HPLC and detected by scintillation counting.

Metabolites of 6-MeC-1,2-diol were produced as follows. A mixture of0.2 mg of 6-MeC-1,2-diol in 0.16 ml dimethyl sulfoxide, 1.28 ml of 0.4 MMgCI2, 1.28 ml of 1.65 M KCI, 0.32 ml of 1.0 M o-glucose-6-phosphate,and 2.56 ml of 0.1 M NADP+ was brought to 5.6 ml with pH 7.4 potassium

phosphate buffer, and 5.6 ml of 9000 x g supernatant from the livers ofAroclor-pretreated rats were added. The resulting mixture was incubatedwith shaking at 37°C for 40 min and then treated with 5 ml acetone.

This mixture was extracted 4 times with 10 ml ethyl acetate. The ethylacetate layers were combined, dried (MgS04), and concentrated to aresidue which was dissolved in MeOH. Metabolites were analyzed byHPLC using System 1. A portion was acetylated as described above for1,2-dihydroxy-6-MeC and analyzed by HPLC.

Metabolism in Vivo. For the metabolism of 6-MeC, five groups of 4

mice each were shaved and, 24 h later, 0.15 ml of an acetone solutionof [3H]6-MeC (1.7 Ci/mmol, 0.07 ¿¿mol)was applied to the shaved back

of each mouse. Mice were sacrificed at time intervals, as indicated inTable 2, and the epidermal metabolites were isolated exactly as described previously (16). Metabolites were analyzed by HPLC, usingSystems 1 and 2. For the comparative study of 5-MeC-1,2-diol and 6-MeC-1,2-diol, 2 groups of 4 mice were shaved and treated with either[3H]5-MeC-1,2-diol (0.05 ¿imol,60 ¿iCi/nmol)or [3H]6-MeC-1,2-diol (0.05

Mmol, 105 fiCi/fimol) per mouse. They were killed 2 h later, and metabolites were isolated and analyzed by HPLC using System 1.

DNA Adducts in Mouse Skin. A group of 21 mice was used. Eachmouse was treated with [3H]6-MeC (0.4 ^mol, 15.2 Ci/mmol) in 0.15 ml

acetone. Twenty-four h later, the mice were sacrificed. DNA was isolated

from the epidermis of the treated areas and hydrolyzed enzymatically todeoxyribonucleosides (17), and the hydrolysates were analyzed by Seph-adex LH-20 chromatography as described previously (16, 18) and by

HPLC using System 3.Bioassay for Tumor-initiating Activity. Each group consisted of 20

female CD-1 mice obtained at age 28-35 days. At age 50-55 days, each

mouse received a single initiating dose of 33 nmol of the appropriatecompound in 0.1 ml acetone. Ten days later, promotion began byapplication of 2.5 /jg 12-O-tetradecanoylphorbol-13-acetate in 0.1 ml of

acetone, 3 times weekly for 20 weeks. Mice were shaved when necessary, and tumors were counted weekly. Statistical significance wasevaluated using the x2 test.

RESULTS

Chart 3 is a chromatogram obtained upon reverse phase HPLCanalysis of the metabolites of 6-MeC formed by incubation with

liver 9000 x g supernatant from rats pretreated with Aroclor1254. This preparation was used for the structural studies because it produces sufficient amounts of metabolites for spectralcharacterization.

The MS of peak 1 had a molecular ion of m/e 276, whichsuggests the presence of dihydrodiol metabolites. From the earlyelution position of peak 1, we suspected that it might contain a

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6-MeC

15 30 45

RETENTION TIME (min)

60

Charts. Chromatogram obtained upon HPLC analysis with System 1 of theethyl acetate extract of an incubation mixture of 6-MeC with cofactors and 9000

x g supernatant from the livers of rats that had been pretreated with Aroclor 1254.The identities of numbered peate are given in the text.

mixture of dihydrodiols with their hydroxyl groups in pseudoaxialconformations. Therefore, it was collected and reanalyzed usingnormal phase HPLC. The resulting chromatogram showed twopeaks, A (88%) and B (12%). The UV spectra of peaks A and Bwere similar to those of 5-MeC-3,4-diol or 5-MeC-9,10-diol and5-MeC-1,2-diol or 5-MeC-7,8-diol, respectively. Peaks A and B

were each collected and treated under conditions known tocause dehydration-of dihydrodiols. Dehydration of peak A gave

a mixture of two chrysenols. One had a UV spectrum similar tothose of 3-hydroxy-5-MeC or 9-hydroxy-5-MeC, and the otherhad a UV spectrum similar to that of 10-hydroxy-5-MeC. Thuspeak A was tentatively identified as 6-MeC-3,4-diol and/or 6-MeC-9,10-diol. Dehydration of peak B gave 7-hydroxy-6-MeC,

which had a UV spectrum and HPLC retention time identical tothose of the synthetic standard. Thus, peak B was identified as6-MeC-7,8-diol. By analogy to other dihydrodiols adjacent toper;'-methyl groups, it is expected to have a frans-diaxial confor

mation (19).The MS of peak 2 of Chart 3 had a molecular ion of m/e 276,

which was consistent with a dihydrodiol. Its UV spectrum wassimilar to that of 5-MeC-1,2-diol. The downfield portion of the

250 MHz NMR spectrum of peak 2 had resonances as follows:52.6 (s, 3H, CH3), 4.3 (bs, 1H, OH), 4.45 (m, 1H, H2), 4.65 (bs,1H, OH), 4.83 (d, 1H, H,, J,^ = 13 Hz), 6.07 (dd, 1H, H3, J3.4=11.3 Hz), 7.1 (dd, 1H, H4, J3.4 = 11.3 Hz), 7.46-7.54 (m, 2H,H8,9),7.75 (s, 1H, H5), 7.8 (d, 1H, H12,J11>12= 9 Hz), 7.86-7.94(m, 1H, H7), 8.47 (d, 1H, H„,J„.12= 9 Hz), and 8.54-8.6 (m,1H, H10).These spectral data are consistent only with 6-MeC-1,2-diol. The structure was confirmed by dehydration to 1-hydroxy-6-MeC, which was identified by comparison of its UV

spectrum and HPLC retention time to those of the syntheticstandard.

Peak 3 was identified as 1,2-dihydroxy-6-MeC by comparison

of its UV spectrum and HPLC retention time to those of the

synthetic standard. Peak 4 was identified as 6-hydroxymethyl-

chrysene by comparison of its UV spectrum, MS, NMR spectrum,and HPLC retention time to those of the synthetic standard.

Peak 5 had a UV spectrum similar to those of 3-hydroxy-5-MeC and 9-hydroxy-5-MeC. It was tentatively identified as 3-and/or 9-hydroxy-6-MeC.

Peak 6 was identified as 1-hydroxy-6-MeC by comparison of

its UV spectrum, MS, NMR spectrum, and HPLC retention timeto those of the synthetic standard. Peak 7 was identified as 7-hydroxy-6-MeC by comparison of its UV spectrum and HPLC

retention time to those of the synthetic standard.The formation of metabolites of [3H]6-MeC was assessed in

liver 9000 x g supernatant from mice treated with either 3-

methylcholanthrene in corn oil or with corn oil alone. The resultsare summarized in Table 1. Data from a previous study of [3H]-5-MeC metabolism carried out under identical conditions areincluded for comparison (20). The metabolism of [3H]5-MeC toits 1,2-dihydrodiol was greater than that of [3H]6-MeC to its 1,2-

dihydrodiol in control mouse liver, but in 3-methylcholanthrene-

pretreated mice these dihydrodiols were formed in similar quantities. Hydroxymethylation of [3H]5-MeC and [3H]6-MeC was

similar in both uninduced and induced mice. Chrysenol formationfrom [3H]5-MeC exceeded that from [3H]6-MeC in both unin

duced and induced mice. The ratio of 1-hydroxy-6-MeC to 7-hydroxy-6-MeC was 1 in control mice but 6.6 in 3-methylchol-anthrene-pretreated mice, reflecting an induction of 1-hydroxy-6-MeC formation and a suppression of 7-hydroxy-6-MeC formation in 3-methylcholanthrene pretreated mice.

The metabolism of [3H]6-MeC was then studied in mouse skin

In vivo, using the technique which we have described previously(16). The radioactivity recovered from mouse epidermis at various intervals after treatment with [3H]6-MeC accounted for

percentages of the dose as follows: 0.67 h (64.4), 1 h (67.9), 2h (47.2), and 4 h (23.2). The ethyl acetate extracts, whichcontained >99% of the recovered radioactivity, were analyzedby HPLC using the identified metabolites as markers. A chromatogram of the organic soluble metabolites formed after 2 h isillustrated in Chart 4. The major metabolite was 6-MeC-1,2-diol.The material eluting in 23-25 min was reanalyzed by normal

phase HPLC and was resolved into two components, corresponding in retention times to 6-MeC-3,4-diol or 6-MeC-9,10-

TablelFormationof selected metabolitesof [3H]6-MeC and [3H]5-MeC by 9000 x g

supernatants from control and 3-methylcholanthrene-treated mice[3H]6-MeC was incubated for 20 min with cofactors and 9000 x g supernatant

from the livers of mice which had been treated with 3-methylcholanthrene in comoil (75 mg/kg) 48 h prior to sacrifice or from control animals treated with com oilonly. Metabolites were analyzed by HPLC as in Chart 3 and identified by coelutionof standards with radioactivity. Data from a previous study of [3H]5-MeC metabo

lism under identical conditions (20) are included for comparison.

&-MeC (nmol/mgprotein)Metabolite1,2-Dihydrodiol

7,8-Dihydrodiol +

3,4(9,1 OHIihydrodiol6(5)-Hydroxymethyl1-Hydroxy7-Hydroxy9- or 3-HydroxyControl0.082

0.130.15

0.0440.0440.043-Meth-

ylcholan-threne0.30

0.210.17

0.0860.0130.0445-MeC

(nmol/mgprotein)Control0.44

0.14a0.20

0.0260.140.223-Meth-ylcholan-

threne0.47

0.22a0.16

0.0940.540.65

" 7,8-Dihydrodiol only.

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METABOLIC ACTIVATION OF 6-MeC AND 5-MeC

25

20

«•'5e

•io

6-MeC-7,8-OIOL6-MeC-3,4/9,IO-DIOLS

6-MeC-I,2-DK)L

l-OH-6-MeC3/9-OH-6-M«C

6-MeC

6 12 18 24 30 36 42 48 54 60 66 72

RETENTION TIME (min)

Chart 4. Chromatogram obtained upon HPLC analysis using System 1 of theethyl acetate extract of mouse epidermis 2 h after treatment with [3H]6-MeC. 6-HOMeC, 6-hydroxymethylchrysene; 1-OH-6-MeC, 1-hydroxy-6-MeC.

\

ta.

1234

TIME I hi

Chart 5. Levels of tritium-labeled metabolites in mouse epidermis at intervalsafter topical application of pHje-MeC: A, 6-MeC-1,2-dtol; A, 6-MeC-7,8-diol and 6-MeC-3,4/9,10-dtols; O, 6-hydroxymethytehrysene; •,total of 6-methylchrysenols.

diol (60%) and 6-MeC-7,8-diol (40%). The Chromatogram illus

trated in Chart 4 was qualitatively similar to that obtained from[3H]5-MeC under essentially identical conditions (16). However,in the metabolism of [3H]5-MeC, the relative amounts of 5-MeC-

1,2-diol and 5-MeC-7,8-diol were similar. Chart 5 summarizesthe formation of metabolites of [3H]6-MeC in mouse skin, at

intervals from 0.33-4 h after treatment. 6-MeC-1,2-diol was the

major ethyl acetate soluble metabolite at each interval, reaching22 pmol/mg epidermis after 2 h. The ratio of 6-MeC-1,2-diol to6-MeC-7,8-diol was 10:1 at the 2-h survival interval. Levels(pmol/mg dry epidermis) of dihydrodiols of [3H]5-MeC formed in

mouse skin 2 h after treatment were: 5-MeC-1,2-diol, 4.5; 5-MeC-7.8-diol, 4.5; 5-MeC-9,10-diol, 2.0 (16).

The further metabolism of 6-MeC-1,2-diol was examined using9000 x g supernatant from the livers of Aroclor-pretreated rats.As illustrated in Chart 6,6-MeC-1,2-diol was efficiently convertedto 1,2-dihydroxy-6-MeC in this system. The metabolite was

identified by comparison of its UV spectrum and HPLC retentiontimes to those of the synthetic standard. The retention times ofthe diacetate derivatives of the metabolite and standard werealso identical. Small amounts of material with retention times thesame as those of 6-MeC-1,2,3,4-tetraols were detected. The

a soRETENTION TIME(min)

75

Chart 6. Chromatograms obtained upon HPLC analysis using System 1 of ethylacetate extracts of incubation mixtures of 6-MeC-1,2-diol with 9000 x g supernatant from the livers of Aroclor-pretreated rats. Peate A and B were identified as 6-MeC-1,2-dtol and 1,2-dihydroxy-6-MeC, respectively. The peaks eluting at 20 and25 min have the same retention times as 6-MeC-1,2,3,4-tetraols but were notpositively identified.

metabolism of 5-MeC-1,2-diol under these conditions was more

complex (data not shown).The metabolism of [3H]6-MeC-1,2-diol and [3H]5-MeC-1,2-diol

was compared in mouse skin in vivo. The HPLC traces obtainedfrom each group of mice were qualitatively similar, showing, inaddition to unmetabolized dihydrodiol, peaks which coeluted withstandard 1,2,3,4-tetraols and 1,2-dihydroxy metabolites as well

as several unidentified metabolites. The extent of formation ofthe identified metabolites from each diol were similar, as follows:(pmol/mg dry epidermis from 6-MeC-1,2-diol and 5-MeC-1,2-diol) 1,2,3,4-tetraol A (1.1 and 1.1); 1,2,3,4-tetraol B (1.4 and1.0); and 1,2-dihydroxy metabolites (1.0 and 0.5).

DNA adduci formation in mouse skin from [3H]6-MeC and[3H]5-MeC was compared. Sephadex LH-20 chromatograms of

deoxyribonucleosides obtained upon enzymatic hydrolysis ofDNA are illustrated in Chart 7. The data for [3H]5-MeC are from

a previous study carried out under essentially identical conditionsand are included for comparison (16). Qualitatively similar Chromatographie profiles were obtained from the two hydrocarbons.Quantitatively there were major differences between the twochromatograms. The formation of early eluting material (40-180ml) from [3H]6-MeC (1.3 pmol/mg DNA) was similar to that from[3H]5-MeC (2.1 pmd/mg DNA). Adducts eluting in the regionfrom 400-700 ml, formed from [3H]5-MeC (3.3 pmol/mg DNA),greatly exceeded those from [3H]6-MeC (0.14 pmol/mg DNA).The adducts eluting from 400-700 ml were reanalyzed by HPLC.The HPLC retention times of the adducts formed from pHJo-MeC were similar to those formed from [3H]5-MeC, which havebeen identified previously as A/2-deoxyguanosine adducts of 5-MeC-1,2-diol-3,4-epoxide and 5-MeC-7,8-diol-9,10-epoxide (18).It is likely that the [3H]6-MeC adducts are also formed from

dihydrodiol epoxides.The tumor-initiating activities of 5-MeC, 6-MeC, 5-MeC-1,2-

diol, and 6-MeC-1,2-diol were compared. The results are summarized in Table 2. In agreement with earlier studies, 5-MeCwas significantly more tumorigenic than was 6-MeC. 5-MeC-1,2-diol was also significantly more tumorigenic than was 6-MeC-1,2-diol.

DISCUSSION

In this study, we sought to determine the reasons why 6-MeCis a weak carcinogen and 5-MeC is a strong carcinogen. The

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METABOLIC ACTIVATION OF 6-MeC AND 5-MeC

24201612841A1A"dpm8004002/

v\/v^^V.400

600ml/

t/\ -A-3100300 500 700 900

36

32

28

24

20

16

12

/v100 300 500 700 900

ELUTION VOLUME ( ml)

Chart 7. Sephadex LH-20 chromalograms of enzymatically hydrolyzed DMAfrom the epidermis of mice treated with (A) [3H]6-MeC and (B) [3H]5-MeC. The data

in (B) are from a previous study (16) and are included for comparison. The specificactivity of [3H]5-MeC used in that study was one-tenth that of the [3H]6-MeC; the

data in (B) were normalized to equal specific activity.

Table 2Comparative tumor-initiating activity on mouse skin of 5-MeC, 6-MeC, 5-MeC-1,2-

diol, and 6-MeC-i,2-diol

Groups of 20 female CD-1 mice were treated with a single dose of 33 nmol of

the appropriate compound in 0.1 ml acetone. Promotion was carried out by threetimes weekly application of 2.5 /ig 12-O-tetradecanoylphorbol-l 3-acetate for 20

weeks.

6-MeC

Control%

of tumor-bearingmice65a-680a"

255Tumors/

mouse1.7

0.052.50.250.05

* P < 0.01 compared to control.

P < 0.01 compared to 6-MeC.c Synthetic and racemte." P < 0.01 compared to 6-MeC-1,2-diol." Metabolically formed; optical purity unknown.

structural difference between the two compounds is the positionof the methyl group. In 5-MeC, the methyl group is in the 4-5

bay region, and this causes an increase in the angles in the bayregion and a distortion from planarity which is not likely to occurin 6-MeC (21, 22). In previous studies we have shown that 5-MeC-1,2-diol, which can form a bay region dihydrodiol epoxide

with the methyl group and an epoxide ring in the same bayregion, is a major proximate carcinogen of 5-MeC and that oneof the dihydrodiol epoxide isomers, anf/-5-MeC-1,2-diol-3,4-

epoxide, is a major ultimate carcinogen (23, 24). It has beensuggested that structural distortions resulting from the presenceof a bay region methyl group in compounds such as 5-MeC and7,12-dimethylbenz[a]anthracene may lead to enhanced formation of potential proximate carcinogenic metabolites such as 5-MeC-1,2-diol (10). The results of the present study demonstrate

that this is not the case in the methylchrysene system. Whereas

5-MeC-1,2-diol formation exceeded 6-MeC-1,2-diol formation in

mouse liver, the opposite result was observed in the targettissue, mouse epidermis. 6-MeC-1,2-diol was the major metabolite of 6-MeC, and its concentration in mouse skin was greaterthan the concentration of 5-MeC-1,2-diol in the skin of micetreated with 5-MeC. Thus, differing extents of formation of 1,2-dihydrodiol metabolites do not account for the greater carcino-genicity of 5-MeC compared to 6-MeC.

The results of the tumorigenicity assay show clearly that 5-MeC-1,2-diol was more tumorigenic than was 6-MeC-1,2-diol.The tumorigenic activity of 6-MeC-1,2-diol was comparable tothat of 5-MeC-7,8-diol (23, 24). 6-MeC-1,2-diol and 5-MeC-7,8-

diol can both form bay region dihydrodiol epoxides, but neitherhas a methyl group in the same bay region as the epoxide ring,as in 5-MeC-1,2-diol. These comparative studies demonstratethat 5-MeC-1,2-diol is a stronger proximate tumorigen than is 6-MeC-1,2-diol.

The results of the comparative DMA binding studies of [3H]-6-MeC and [3H]5-MeC are consistent with the lower tumorigenic

activity of 6-MeC-1,2-diol than of 5-MeC-1,2-diol. Major DNAadducts are formed from 5-MeC, through its 1,2-dihydrodiol-3,4-epoxide metabolites. Minor adducts are formed from its 7,8-dihydrodiol-9,10-epoxides (16, 18). Radioactive peaks, with retention times on Sephadex LH-20 and HPLC similar to these 5-MeC dihydrodiol epoxide adducts, were also formed from [3H]6-

MeC but in much lower concentrations. These adducts areassumed to be 6-MeC dihydrodiol epoxide-DNA adducts, andtheir levels in mouse skin were approximately 1/2oas great as thecorresponding 5-MeC dihydrodiol epoxide-DNA adducts. Thus,although 6-MeC-1,2-diol was formed to a greater extent inmouse skin than was 5-MeC-1,2-diol, its tumorigenicity andconversion to apparent dihydrodiol epoxide-DNA adducts wasfar less than that of 5-MeC-1,2-diol.

We sought to explain these differences by studying the furthermetabolism of 6-MeC-1,2-diol. Our initial experiments were carried out using liver 9000 x g supernatant from Aroclor-pretreatedrats, in order to facilitate identification of metabolites. In thissystem, 6-MeC-1,2-diol underwent a remarkably smooth conversion to 1,2-dihydroxy-6-MeC, as illustrated in Chart 6. Onlysmall amounts of material with the retention times of 6-MeC-1,2,3,4-tetraols, which would be formed by hydrolysis of 6-MeC-1,2-diol-3,4-epoxide, were evident. The smooth conversion of 6-MeC-1,2-diol to 1,2-dihydroxy-6-MeC, which was not observedin the metabolism of 5-MeC-1,2-diol under the same conditions,

seemed to offer an explanation for the relatively low tumorigenicactivity of 6-MeC-1,2-diol. However, the results of our comparative studies of [3H]6-MeC-1,2-diol and [3H]5-MeC-1,2-diol me

tabolism in mouse skin indicated that there were not majordifferences in the target tissue metabolism of these two dihydro-diols. Both dihydrodiols were converted to tetraols and 1,2-dihydroxy metabolites, as well as to several unidentified metabolites, to apparently similar extents. These results demonstratethe importance of target tissue metabolism studies in investigations of carcinogen activation and suggest that the difference inactivity between 5-MeC-1,2-diol and 6-MeC-1,2-diol may not be

due to differing extents of conversion to dihydrodiol epoxides.In previous studies we have shown that anf/-5-MeC-1,2-diol-

3,4-epoxide is a stronger tumorigen than is anf;-5-MeC-7,8-diol-9,10-epoxide (24) and that it binds to DNA to a greater extentthan does anf;-5-MeC-7,8-diol-9,10-epoxide (25). These studies

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METABOLIC ACTIVATION OF 6-MeC AND 5-MeC

led to the conclusion that the enhancing effect of a bay regionmethyl group on methylchrysene tumorigenicity was due to theunique tumorigenicity and greater DMA binding properties of adihydrodiol epoxide isomer with the methyl group and epoxidering in the same bay region. The results of the present study arein agreement with this hypothesis. They lead to the predictionthat the DNA binding properties of anf/-6-MeC-1,2-diol-3,4-epox-ide would be low compared to those of anf/-5-MeC-1,2-diol-3,4-

epoxide. This prediction is currently being tested experimentally.The structural requirements favoring tumorigenicity of meth

ylated alternant PAH are the presence of a bay region methylgroup and free pen-position both adjacent to an unsubstitutedangular ring, as in 5-MeC (4, 5). In the methylchrysene series,only 5-MeC fulfills these requirements and can form a bay regiondihydrodiol epoxide with the methyl group and epoxide ring inthe same bay region. The results of this study provide furtherevidence for the importance of this unique type of bay regiondihydrodiol epoxide in methylchrysene tumorigenesis and probably in methylated PAH tumorigenesis.

ACKNOWLEDGMENTS

We thank George Baiamkasand Dr. NalbandHussain for their assistance in themetabolismexperiments, the members of the ResearchAnimalFacility for carryingout the btoassay. and Lori DeMarco and Gail Thiede for typing and editing themanuscript.

REFERENCES

1. Hecht, S. S., Sondinoli,W. E., and Hoffmann, D. Chemicalstudies on tobaccosmoke. XXIX. Chrysene and methylchrysenes: presence in tobacco smokeand carcinogenicity.J. Nati. Cancer Inst., 53. 1121-1133,1974.

2. Hecht, S. S., Loy, M., Maronpot, R. R., and Hoffmann, D. Comparativecarcinogenicityof 5-methylchrysene,benzo[a]pyrene,and modifiedchrysenes.Cancer Lett., 1: 147-154,1976.

3. Dippte,A., Moschel, R. C., and Bigger, C. A. H. Polynucleararomatic hydrocarbons. In: C. E. Searle (ed.), Chemical Carcinogens, Ed. 2, pp. 41-163.Washington, DC: AmericanChemical Society, 1984.

4. Hecht, S. S., Amin, S., Melikian.A. A., LaVoie, E. J., and Hoffmann, D. Effectsof methyl and fluorinesubstitution on the metabolicactivation and tumorigenicity of polycyclic aromatic hydrocarbons. ACS Symposium Series, in press,1985.

5. Hecht, S. S., Amin, S., Rivenson,A., and Hoffmann, D. Tumor initiatingactivityof 5,11-dimethylchryseneand the structural requirements favoring carcinogenicity of methylated polynuclear aromatic hydrocarbons. Cancer Lett., 8: 65-70, 1979.

6. LaVoie, E. J., Bedenko,V., Tulley-Freiler,L, and Hoffmann,D. Tumor initiatingactivity and metabolism of polymethylated phenanthrenes. Cancer Res., 42:4045-4049, 1982.

7. Wistocki, P. G., Fiorentini,K. M., Fu, P. P., Yang, S. K., and Lu, A. Y. H. Tumorinitiating activities of the twelve monomethylbenz[a]anthracenes.Carcinogen-esis (Lond.),3: 215-217, 1982.

8. Coombs, M. M, Bhatt, T. S., and Croft, C. J. Correlation between carcinoge

nicity and chemicalstructure in cyctopenta[a]phenanthrenes.Cancer Res.,33:832-837, 1973.

9. Iyer, R. P., Lyga, J. W., Secrist, J. A. Ill, Daub, G. H., and Slaga, T. J.Comparativetumor initiatingactivity of methylatedbenzo[a]pyrenederivativesin mouse skin. Cancer Res., 40:1073-1076,1980.

10. DiGiovanni,J., Diamond, L., Harvey, R. G., and Slaga, T. J. Enhancementofthe skin tumor initiatingactivity of polycyclicaromatichydrocarbonsby methylsubstitution at non-benzo bay-region positions. Carcinogenesis (Lond.), 4:403-407,1983.

11. Levin,W., Wood, A. W., Chang,R. L., Newman,M. S.,Thakker, D. R., Conney,A. H., andJerina, D.M. The effect of steric strain in the bay region of polycyclicaromatic hydrocarbons: tumorigenicity of alkyl-substituted benzofajanthra-cenes. Cancer Lett., 20: 139-146,1983.

12. Heidelberger,C., Baumann, M. E., Greisbach, L., Ghobar. A., and Vaughan,T. M. The carcinogenic activities of various derivatives of dibenzanthracene.Cancer Res., 22: 78-83,1962.

13. Pataki, J., Lee, H., and Harvey, R. G. Carcinogenic metabolites of 5-methylchrysene. Carcinogenesis(Lond.),4: 399-402,1983.

14. Hecht, S. S., LaVoie, E., Mazzarese,R., Amin, S., Bedenko,V., and Hoffmann,D. 1,2-Dihydro-1,2-dihydroxy-5-methylchrysene,a major activated metaboliteof the environmental carcinogen, 5-methylchrysene.Cancer Res., 38: 2191-2194, 1978.

15. Lock, G., and Schneider,R. Chloromethylationof naphthalene.III. 1-Methyl-4-halomethyl- and 1,4-bis (bromomethyl)naphthalene.Chem. Ber., 91: 1770-1774,1958.

16. Melikian,A. A., LaVoie,E. J., Hecht,S. S., and Hoffmann,D. 5-Methylchrysenemetabolismin mouse epidermisin vivo, diol epoxide-DNAadduci persistence,and diol epoxide reactivity with DNA as potential factors influencing thepredominance of 5-methylchrysene-1.2-diol-3.4-epoxide-DNA adducts inmouse epidermis. Carcinogenesis(Lond.),4: 843-849, 1983.

17. Baird, W., and Brookes, P. Isolation of the hydrocarbon-deoxyribonucleosideproducts from the DNA of mouse embryo cells treated in culture with 7-methylbenz[a]anthracene-3H.Cancer Res., 33: 2378-2385,1973.

18. Melikian, A. A., Amin, S., Hecht, S. S., Hoffmann, D., Pataki, J., and Harvey,R. G. Identificationof the major adducts formed by reaction of 5-methylchrysene an(/-dihydrodiol epoxktes with DNA in vitro. Cancer Res., 44: 2524-2529,1984.

19. Yang,S. K., Chou, M. W., and Fu, P. P. Metabolicand structural requirementsfor the carcinogenic potencies of unsubstituted and methyl-substituted polycyclic aromatic hydrocarbons. In: B. Pullmann,P. O. P. Ts'o, and H. Gelboin

(eds.),Carcinogenesis:Fundamentalmechanismsand EnvironmentalEffects,pp. 143-156. London: D. Reidel, 1980.

20. Amin, S., Camanzo, J., and Hecht, S. S. Inhibition by a peri-fluorine atom of1,2-dihydrodiolformation as a basis for the lower tumorigenicity of 12-fluoro-5-methylchrysene than of 5-methylchrysene. Cancer Res., 44: 3772-3778,1984.

21. Kashino,S., Zacharias,D. E.. Prout, C. K., Carre«,H. L., Glusker,J. P., Hecht,S. S., and Harvey, R. G. Structure of 5-methylchrysene,Cu>H,4.Acta Cryst,040:536-540,1984.

22. Zacharias, D. E., Kashino, S., Glusker, J. P., Harvey, R. G., Amin, S., andHecht, S. S. The bay region geometry of some 5-methylchrysenes: stericeffects in 5,6- and 5,12-dimethylchrysenes.Carcinogenesis(Lond.), 5: 1421-1430,1984.

23. Hecht, S. S., Rivenson, A., and Hoffmann, D. Tumor-initiating activity ofdihydrodiols formed metabolically from 5-methylchrysene. Cancer Res., 40:1396-1399,1980.

24. Hecht, S. S., Radok, L., Amin, S., Hu«,K., Melikian, A. A., Hoffmann, D.,Pataki, J., and Harvey, R. G. Tumorigenicityof 5-methylchrysenedihydrodiolsand dihydrodiol epoxides in newborn mice and on mouse skin. Cancer Res.,45: 1449-1452,1985.

25. Melikian, A. A., Leszczynska, J. M., Amin, S., Hecht, S. S., Hoffmann, D.,Pataki, J., and Harvey, R. G. Rates of hydrolysis and extents of DNA bindingof 5-methylchrysenedihydrodiolepoxides.CancerRes.,45:1990-1996,1985.

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1985;45:6406-6412. Cancer Res   Shantu Amin, Keith Huie, Assieh A. Melikian, et al.   5-MethylchryseneCarcinogen 6-Methylchrysene and the Strong Carcinogen Comparative Metabolic Activation in Mouse Skin of the Weak

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