[CANCER RESEARCH 42, 4779-4783, November 1982]0008-5472/82/0042-OOOOS02.00

Communication

Metabolism and Tumorigenicity of 7-, 8-, 9-, and 10-Fluorobenzo(a)pyrenes

Donald R. Buhler,1 Figen Unlu,2 Dhiren R. Thakker, Thomas J. Slaga, Melvin S. Newman,Wayne Levin, Allan H. Conney, and Donald M. Jerina3

Laboratory ol Bioorganic Chemistry, NIADDK, NIH, Bethesda, Maryland 20205 ID. R. B., F. U., D. R. T.. D. M. J.]; Biology Division, Oak Ridge National Laboratory,Oak Ridge, Tennessee 3 7803 [T. J. S.]: Chemistry Department, The Ohio State University, Columbus, Ohio 43210 [M. S. N.¡;and Department of Biochemistry andDrug Metabolism, Hoffman-La Roche Inc. Nutley. New Jersey 07110 ¡W.L., A. H. C.]

ABSTRACT

The skin tumor-initiating activities of 7-, 8-, 9-, and 10-

fluorobenzo(a)pyrenes have been compared to that ofbenzo(a)pyrene in female Sencar mice after 16 weeks of promotion with 12-O-tetradecanoylphorbol-13-acetate. Single in

itiating doses of 200 or 400 nmol of each hydrocarbon weretested, and the mice were treated twice weekly with 3.2 nmolof the promoter. Under these conditions, benzo(a)pyrenecaused an average of 2.9 and 5.7 papillomas/mouse, respectively, whereas none of the four fluorinated hydrocarbons hadsignificant tumor-initiating activity. Examination of the hepaticmetabolism of 7- and 8-fluorobenzo(a)pyrene revealed that a7,8-dihydrodiol was not detected as a metabolite; thus, thebay-region diol-epoxide pathway known to be responsible forthe tumorigenic activity of benzo(a)pyrene is blocked. Although7,8-dihydrodiols are formed from 9- and 10-fluorobenzo(a)-

pyrene, these dihydrodiols with fluorine substituted on the9,10-double bond may not be converted to diol-epoxides bythe cytochrome P-450 system, or such fluorinated 7,8-diol-9,10-epoxides may not be tumorigenic.

INTRODUCTION

BP" was the first member of the polycyclic aromatic hydro

carbon class of carcinogens for which proximate and ultimatecarcinogens were identified (3, 20, 24, 25). The metabolicpathway to the chemically reactive species responsible for theadverse biological activity of BP consists of cytochrome P-450-catalyzed formation of (+)-benzo(a)pyrene (7fi,8S)-oxide

which is then hydrated by microsomal epoxide hydrolase to(-)-irans-(7fî,8fî)-dihydroxy-7,8-dihydrobenzo(a)pyrene andsubsequently epoxidized by the cytochrome P-450 systemto ( + )-7/?,8a-dihydroxy-9a,10a-epoxy-7,8,9,10-tetrahydro-benzo(a)pyrene, the (7f?,8S)-diol-(9S,10n)-epoxide in whichthe benzylic 7-hydroxyl group and epoxide oxygen are trans.

The final epoxidation step is also known to be catalyzed byother enzyme systems (4, 27). The above-cited tumor studieswere paralleled by metabolism (37, 48), DNA binding (21, 36,

' We are grateful to the Environmental Health Science Center, Oregon StateUniversity, Corvallis for providing salary support to this author for a 1-year

sabbatical leave at the NIH.2 Present address: Turkish Atomic Energy Commission, Ankara, Turkey.3 To whom requests for reprints should be addressed.4 The abbreviations used are: BP, benzo(a)pyrene; HPLC, high-performance

liquid chromatography; 7-FBP, 7-fluorobenzo(a)pyrene; 8-FBP, 8-fluoro-benzo(a)pyrene; 9-FBP, 9-fluorobenzo(a)pyrene; 10-F8P, 10-fluorobenzo(a)-pyrene; 6-FBP, 6-fluorobenzo(a)pyrene; TPA, 12-O-tetradecanoyl-phorbol-13-acetate: BP 7-8-dihydrodiol. frans-7,8-dihydroxy-7,8-dihydrobenzo(a)pyrene.

Received May 24, 1982; accepted August 6, 1982.

40), and mutagenesis (13, 30, 41, 43, 46) experiments whichimplied the same conclusions.

On the basis of the above biological observations and avariety of chemical considerations, we proposed (16-18) thatrelated bay-region diol-epoxides would be prime candidates as

ultimate carcinogens from other polycyclic aromatic hydrocarbons and that aryl ring substitutents, which are known to inhibitthe monooxygenase-catalyzed epoxidation of formal aromaticdouble bonds to which they are attached (16-18), on the

critical benzo ring might cause the observed decreases intumorigenic response by retardation of the diol-epoxide path

way. From a mechanistic standpoint, both electronic and stericfactors could cause a decreased rate of cytochrome P-450-

catalyzed epoxidation at a substituted aromatic double bond.In general, fluorine and methyl groups have been the mostextensively used substituents to probe the tumorigenic activityof the polycyclic aromatic hydrocarbons (cf. Refs. 16 to 18).The present study describes the metabolism and tumorigenicityof the 4 possible monofluorinated BP derivatives on the 7,8,-9,10-benzo ring.

MATERIALS AND METHODS

Chemicals. BP, purchased from Aldrich Chemical Co., Milwaukee,Wis., was essentially pure based on the HPLC conditions used to studyits metabolism. 7-FBP was obtained by thermal decomposition of the

diazonium fluoroborate at the 7 position of BP (see Ref. 33, Footnotes2 and 3). A combination of HPLC (Du Pont Zorbax ODS column elutedwith acetonitrile), open-column chromatography (Florisil eluted se

quentially with méthylènechloride and then 10% hexane in acetone),and recrystallization (cyclohexane-benzene) provided material withm.p. 178.1 -178.6 (corrected). Material so purified was identical to 7-

FBP obtained by an alternate, more efficient synthesis (33). Samplesof 8- and 9-FBP (31) as well as 10-FBP (32) were obtained via

unequivocal chemical synthesis. All 4 fluorinated derivatives werejudged to be free of BP and essentially homogeneous on the basis ofthe HPLC conditions used to study their metabolism. The '9F-NMR

spectra (recorded in deuteriochloroform with internal C6F6 on a 100-

MHz Jeol spectrometer) gave no indication that any of the samplescontained other fluorinated isomers: 7-FBP, F7 centered at 40.38 ppmas a doublet of doublets; 8-FBP, F8 centered at 46.88 ppm as amultiplet; 9-FBP, F9 centered at 48.94 ppm as a multiplet; and 10-FBP,

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D. R. Buhler et al.

Fio centered at 52.47 ppm as doublet of multiplets. The 6-FBP used

for comparison purposes in the present study was free of BP but wasof slightly lower purity (several minor peaks totaling ~5%) than thatdescribed previously,5 on the basis of analysis by HPLC with the

column effluent monitored at 254 nm.Incubation Conditions. Microsomes were prepared as described

from the livers of 3-methylcholanthrene-treated immature male rats ofthe Long-Evans strain (26) and contained 1.3 nmol cytochrome P-450

per mg of protein, as determined spectrally (34). Incubation mixturescontained 25 mg of microsomal protein, 5 mmol of potassium phosphate buffer (pH 7.4), 0.15 mmol of MgCI2, 25 ¡imcAof NADPH, and4.0 /¿molof substrate in a final volume of 50 ml. Substrates were addedin acetone such that the final acetone concentration was 5%. Incubations were for 10 min at 37°and were terminated by extraction of the

substrates and products into one volume of acetone and 2 volumes ofethyl acetate. The organic phase was separated, dried over anhydroussodium sulfate, and concentrated by rotary evaporation under vacuum.The residues were dissolved in methanol for analysis by HPLC.

Separation and Characterization of Metabolites. Metabolites wereseparated by HPLC on a Spectra Physics 3500B liquid Chromatographequipped with a DuPont Zorbax ODS column (6.2 mm x 25 cm). Onemin after injection, the column was eluted with a linear gradient of 60to 99% methanol in water for 40 min at a constant flow rate of 1.2 ml/min at which point elution with 99% methanol was continued for 20min. The column eluent was monitored at 254 nm. For purposes ofchemical characterization of metabolites, samples were also chromat-

ographed on a DuPont Zorbax SIL HPLC column (6.2 mm x 25 cm)eluted with cyclohexane:dioxane:ethanol (87.8:12:1.2), and peakswere cross-compared to the ODS separations on the basis of their UV

spectra. All peaks in the metabolite regions, which constituted about5% or more of the total detector response, were collected by combinations of chromatography on the 2 columns.

Identification of metabolites was achieved by comparison of their UVspectra in methanol with positionally related metabolites formed fromBP and 6-FBP.5 Peaks were identified as containing phenols only when

characteristic bathochromic shifts were observed in the presence ofalkali. Peaks characterized as dihydrodiols had UV spectra virtuallyidentical to those positional isomers from BP and 6-FBP. As notedpreviously,5 the presence of a 6-fluoro substituent has practically no

effect on the UV spectra of several phenols and dihydrodiols of BP.Additional support for assignments of phenols and dihydrodiols wasbased on their mass spectra. Chemical ionization mass spectra withammonia gas gave m/e = 287 (M* + 1 - H2O) and with NO-N2 gas

gave m/e = 305 for fluorinated dihydrodiols. Fluorinated phenols gavethe expected (M + + 1) and (IvT) with ammonia gas and NO-N2 gas,

respectively. Due to higher polarity, dihydrodiols always eluted prior tophenols on the ODS column. Metabolites characterized as quiñonesare only tentative identifications and are based on expected molecularweights and/or broad, weak maxima in the 220- to 500-nm region

(35).Tumor Studies. Female Sencar mice, originally obtained from Dr.

R. K. Boutwell at the University of Wisconsin, Madison, are presentlyraised at Oak Ridge, Tenn. Mice 7 to 9 weeks of age were shaved withsurgical clippers 2 days before treatment, and only those animals inthe resting stage of the hair cycle were used. Groups of 30 micereceived a single topical application of 200 or 400 nmol of BP orfluorobenzo(a)pyrene dissolved in 0.2 ml of spectroquality acetoneunder subdued light, followed 1 week later by twice-weekly applications

of 3.2 nmol of TPA in 0.2 ml of acetone. TPA was obtained fromChemical Carcinogenesis Inc., Eden Prärie, Minn. The incidence ofpapillomas was recorded weakly. Mice were selected at random forhistological verification of the tumors.

Table 1Skin tumor-initiating activity of BP and 5 of its isomerie monotluorinated

derivatives in Sencar mice after 16 weeks of promotion by TPA

Thirty female Sencar mice received a single topical application of compoundin 200 fil of acetone. One week later, the mice received twice-weekly topicalapplications of TPA (3.2 nmol) in 200 /xl of acetone. From 28 to 30 mice in eachexperimental group were alive at the end of 16 weeks of tumor promotion.

CompoundBP6-FBP7-FBP8-FBP9-FBP1

0-FBPBP

only"TPAonlycDose2004002004002004002004002004002004004003.2%

of mice withpapillomas6790435013101070007010Papillomas/mouse2.90

±0.52a5.70

±0.580.53±0.120.83±0.210.13±0.050.13±0.080.10±0.020.07±0.050000.07

±0.0200.10

±0.05

Mean ±S.E.BP tumor initiation only.

c TPA promotion only.

RESULTS AND DISCUSSION

The skin tumor-initiating activity of BP and its 6-, 7-, 8-, 9-,and 10-monofluoro derivatives are compared at 200- and 400-nmol initiating doses in Table 1. The 7-, 8-, 9-, and 10-substi-tuted BPs lacked significant initiating activity at either of thedoses tested under conditions where BP was quite active andgave 2.9 and 5.7 papillomas/mouse, respectively. As described previously, 6-FBP has significant tumor-initiating activity but is less active than BP on mouse skin.5-6

In an attempt to find a basis for the virtual lack of tumor-initiating activity of the 4 benzo ring-fluorinated BPs, theirmetabolism was examined with hepatic microsomes from 3-methylcholanthrene-treated rats. Reversed-phase HPLC metabolism profiles for these substrates as well as BP and 6-FBPare shown in Chart 1. Since radioactive substrates were unavailable for the benzo ring derivatives, the emergence of metabolites from the column for the 6 substrates was monitoredby absorbance at 254 nm. Although this method of analysis ofmetabolites is purely qualitative, interestingly the ratio of peakarea based on absorbance at 254 nm to radioactivity formetabolites from [3H]6-FBP varied by less than a factor of 2

throughout the profile (see Footnote 5). Thus, the presence ofsignificant metabolites should be detected by their absorbanceat 254 nm.

Interpretation of the results in Chart 1 requires an understanding of the possible fate of an arene oxide with fluorinesubstituted on the oxirane ring. If such an arene oxide is formedby the cytochrome P-450 system and if it is hydrated to adihydrodiol by epoxide hydrolase, the resultant fluorinateddihydrodiol would be expected to spontaneously break downto a catechol which might further autoxidize to a quinone (Chart2). Thus, fluorine substitution at the 7 or 8 position shouldpreclude isolation of a 7,-8-dihydrodiol. It is noteworthy thatthe 7,8-quinone of BP could not be detected as a metabolite of

5 D. R. Buhler, F. Unlu, D. R. Thakker. T. J. Slaga. A H. Conney, A. W Wood,W. Levin, and D. M. Jerina, Effect of a 6-fluoro substituent on the metabolismand biological activity of benzo(a)pyrene. submitted for publication.

6 M. K. Buening. W. Levin, A. W. Wood, R. L. Chang. I. Agranat, D. R. Buhler.

H. D. Mah. O. Hernandez, D. M. Jerina, A. H. Conney, E. C. Miller, and J. A.Miller. Fluorine substitution as a probe for the role of the 6-position ofbenzo(a)pyrene in Carcinogenesis, submitted for publication.

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Fluorinated BPs

BP 6-FBP

30

TIME (min)30

TIME (min)

7-FBP 8-FBP

3OTIME (min)

30TIME (min)

9-FBP 10-FBP

4,5-DIHYDRODIO

30TIME (min)

30TIME (min)

Chart 1. Comparison of the metabolism profiles obtained from BP and 5 of its monofluorinated derivatives. HPLC conditions are as described in "Materials andMethods" as is the spectral basis of the structural assignments . When all 6 hydrocarbons were chromatographed simultaneously, BP, 8-FBP. and 9-FBP were

essentially cochromatographic. whereas 7-FBP. 10-FBP, and 6-FBP had 3.9, 5.9, and 8.9% longer retention times, respectively.

7-FBP under Chromatographie conditions, which separate itfrom the 1,6- and 3,6-quinones. Thus, for 7-FBP, either thefluorinated 7,8-oxide is not formed or if formed is not hydrated

by epoxide hydrolase. Similarly, fluorine at the 9 or 10 position

should preclude isolation of a 9,10-dihydrodiol. As seen from

Chart 1, such is the case. The expected 2 dihydrodiols wereformed from 7-FBP (4,5- and 9,10-dihydrodiols), 9-FBP (7,8-and 4,5-dihydrodiols), and 10-FBP (4,5- and 7,8-dihydrodiols).

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Chart 2. Expected fate of a fluorine-substituted dihydrodiol formed from afluorine-substituted arene oxide. Carbon atoms substituted by both a hydroxylgroup and a halogen atom rapidly break down into hypoxanthine and thecorresponding carbonyl compound; in this case, a keto tautomer of a catechol.

Only the 4,5-dihydrodiol was detected from 8-FBP. The reasonfor the absence of a 9,10-dihydrodiol in this profile is unknown.The absence of a 7,8-dihydrodiol in the metabolite profile from7-FBP and 8-FBP and of a 9,10-dihydrodiol from 9-FBP and10-FBP may be due to decomposition but is more probably

due to a markedly decreased rate of formation of the appropriate arene oxide precursor relative to the same ring positionsin BP. The presence of a halogen substituent on a double bondis known to retard its rate of chemical epoxidation, generallyby more than an order of magnitude (28). The metabolite peakslabeled as phenols from 7-, 8-, 9-, and 10-FBP had UV spectravery similar to 3-hydroxybenzo(a)pyrene, as was the case forthe major phenol from 6-FBP.5 No attempts were made to

further characterize phenols or quiñones. In terms of tumori-genie response, lack of formation of 7,8-dihydrodiols from 7-and 8-FBP precludes the formation of bay-region 7,8-diol-9,10-epoxides and thus explains their inactivity. For both 9- and 10-FBP, 7,8-dihydrodiols are formed but have a fluorine substituent on their 9,10-double bonds. Although little is known aboutthe cytochrome P-450-catalyzed epoxidation of fluorine-sub

stituted double bonds or the reactivity of the resulting epoxides,it is probable that such double bonds are only very slowlyoxidized by this system. If 7,8-diol-9,10-epoxides are formed,

the fluorinated epoxide group would probably undergo internalrearrangements in preference to reaction with external nucleo-

philes, as required in the expression of tumorigenic activity bychemicals (29). Examples of epoxides with a single fluorinesubstituent do not seem to be known. A single chlorine orbromine substituent is known to markedly enhance reactivityof epoxides via rearrangement processes (23, 28). a-Chloro-styrene oxide, for example, violently rearranges to chloroace-

tophenone and chlorophenylacetaldehyde (28). Although otherexplanations are possible for the inactivity of 9- and 10-FBPon mouse skin, decreased reactivity of a fluorine-substituteddouble bond toward epoxidation and poor reactivity of fluorine-substituted epoxides toward nucleophiles are probably majorcontributors to the lack of tumorigenic activity of 9- and 10-

FBP. It is particularly noteworthy that substitution of a methylgroup at the 9 position of the 7,8-dihydrodiol of BP dramatically

blocks tumorigenic activity (15).The extent to which a dihydrodiol is metabolized to a diol-

epoxide is a probable factor in determining overall tumorigenicresponse, and dihydrodiol conformation can be an importantfactor in determining the degree to which such metabolismoccurs (cf. Refs. 6, 19, and 38). For example, the predominantly pseudo-diequatorial 7,8-dihydrodiol of BP is converted

almost entirely to bay-region 7,8-diol-9,10-epoxides with livermicrosomes from 3-methylcholanthrene-treated rats, whereas

such products account for a smaller percentage of the totalmetabolites from the pseudodiaxial 7,8-dihydrodiol of 6-FBP.5

Whether or not a 9-fluoro substituent on the 7,8-dihydrodiol of

BP would cause a related conformational change is presentlyunknown. (See "Note Added in Proof").

The ability of rat liver microsomes to metabolically activate7-, 8-, 9-, and 10-FBP to products mutagenic to Salmonella

typhimuhum strains TA98 and TA1538 has been examinedwith the result that metabolite(s) of 8-FBP produced moremutations than did metabolites of BP or the other 3 fluorinatedisomers (8). Studies with a reconstituted system containingcytochrome P-450c and varying amounts of homogeneousepoxide hydrolase have indicated that the 4,5-oxide of BP

accounts for much of the mutagenic activity caused by BP inthe presence of liver microsomes (45). Since the 4,5-dihydrodiol was the only dihydrodiol identified from 8-FBP, the 4,5-oxide of 8-FBP represents a very substantial metabolite fromthis hydrocarbon and may account for the enhanced mutagen-icity of 8-FBP relative to BP and the other fluorinated isomers.

The present results provide strong support for (+)7/S,8a-dihydroxy-9a,10a-epoxy-7,8,9,10-tetrahydrobenzo(a)pyrene

as the most important ultimate carcinogen formed from BP andfor the bay-region theory (16-18). The observed inactivity of7-, 8-, 9-, and 10-FBP is generally paralleled by studies on

several other fluorinated hydrocarbons (cf. Refs. 16 to 18 and1, 2, 5, 10, 11, 14, and 44). Methyl substituents on the criticalbenzo ring which forms the bay-region of polycyclic aromatic

hydrocarbons seem to play a role similar to that of fluorine inretarding tumorigenic activity (cf. Refs. 16 to 18 and 12 and15). In contrast to fluorine as a substituent, dihydrodiols witha methyl substituent at a carbinol position have been detectedas metabolites (42, 47). Although these were only qualitativestudies, it was clear that these methyl-substituted dihydrodiols

represented a much smaller percentage of total metabolitesthan did the same dihydrodiol from the unsubstituted hydrocarbon. Although one s.c. study found high activity for 7-methylbenzo(a)pyrene, the doses chosen were too high to tellwhether the methyl substituted BP had significant activity whencompared to BP (9). Additionally, the s.c. tumor model issomewhat unusual (22) in that results obtained with it oftenconflict with results obtained on skin and in other tumor models(7, 15, 39).

Noted Added in ProofIn a companion study on the metabolism and tumorigenicity of 6-FBP,5 we

have found that the 7,8-dihydrodiol of 6-FBP adopts the unusual pseudo-diaxialconformation (J78 = 3.5 Hz in CDCI3) due to the presence of the peri 6-fluorosubstituent. The pseudo-diequatorial 7.8-dihydrodiol of BP has JTB = 10.4 Hz.Dr. Uma Kapur of the NIH has now isolated a sufficient amount of the 7,8-dihydrodiol of 9-FBP to obtain an nmr spectrum and has found Jr.a = 7.2 Hz,indicative that this dihydrodiol has roughly equal populations of both conformations. In contrast, a 9-methyl substituent on the 7,8-dihydrodiol (15) does notaffect the conformation (J7.e =11 Hz).

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