Effects of a 6-Fluoro Substituent on the Metabolism of Benzo(a)pyrene 7,S-Dihydrodiol to Bay-region Diol Epoxides by Rat Liver Enzymes*

Metabolism of fru~-?,S-dihydroxy-7,8-dihydr0-6fluorobenzo(c6)pyrene by liver microsomes from 3methylcholanthrene-treated rats and by a highly purified monooxygenase system, reconstituted with cytochrome P-450~ has been examined. Although both the  fluorinated  and  unfluorinated ?$-dihydrodiol formed from benzo(a)pyrene by liver microsomes share ( R , ~ ) a ~ l u t e c o n ~ ~ r a t i o n , the fluorinated dihydrodiol prefers the conformation in which the hydroxyl groups are pseudodiaxial due to the proximate fluorine. The fluorinated 4,6and 9,lO-dihydrodiols are also >97% the (R,R)-enantiomers. For benzo(a)pyrene, metabolism of the (?R,8R)-dihydrodiol to a bay-region 7,8-diol-9,10-epoxide in which the benzylic hydroxyl group and epoxide oxygen are trans constitutes the only known pathway to an ultimate carcinogen. With the microsomal and the purified mon~xygenase system, this pathway accounts for 7642% of the total metabolites from the 7,s-dihydrodiol. In contrast, only 32-49% of the corresponding diol epoxide is obtained from the fluorinated dihydrodiol and this fluorinated diol epoxide has altered conformation in that its hydroxyl groups prefer to be pseudodiaxial. Much smaller amounts of the diastereomeric 7,8-diol-S,lO-epoxides in which the benzylic hydroxyl groups and the epoxide oxygen are cis are formed from both dihydrodiols. As the fluorinated diol epoxides are weaker mutagens toward bacteria and mammalian cells relative to the unfluorinated diol epoxides, conformation appears to be an important determinant in modulating the biological activity of diol epoxides. One of the more interesting metabolites of 6-fluorinated 7,s-dihydrodiol was a relatively stable arene oxide, probably the 4,5-oxide, which is resistant to the action of epoxide hydrolase.

Metabolism of fru~-?,S-dihydroxy-7,8-dihydr0-6fluorobenzo(c6)pyrene by liver microsomes from 3methylcholanthrene-treated rats and by a highly purified monooxygenase system, reconstituted with cytochrome P-450~ has been examined. Although both the fluorinated and unfluorinated ?$-dihydrodiol formed from benzo(a)pyrene by liver microsomes share ( R ,~) -a~l u t e c o n~~r a t i o n , the fluorinated dihydrodiol prefers the conformation in which the hydroxyl groups are pseudodiaxial due to the proximate fluorine. The fluorinated 4,6-and 9,lO-dihydrodiols are also >97% the (R,R)-enantiomers. For benzo(a)pyrene, metabolism of the (?R,8R)-dihydrodiol to a bay-region 7,8-diol-9,10-epoxide in which the benzylic hydroxyl group and epoxide oxygen are trans constitutes the only known pathway to an ultimate carcinogen. With the microsomal and the purified mon~xygenase system, this pathway accounts for 7642% of the total metabolites from the 7,s-dihydrodiol. In contrast, only 32-49% of the corresponding diol epoxide is obtained from the fluorinated dihydrodiol and this fluorinated diol epoxide has altered conformation in that its hydroxyl groups prefer to be pseudodiaxial. Much smaller amounts of the diastereomeric 7,8-diol-S,lO-epoxides in which the benzylic hydroxyl groups and the epoxide oxygen are cis are formed from both dihydrodiols. As the fluorinated diol epoxides are weaker mutagens toward bacteria and mammalian cells relative to the unfluorinated diol epoxides, conformation appears to be an important determinant in modulating the biological activity of diol epoxides. One of the more interesting metabolites of 6-fluorinated 7,s-dihydrodiol was a relatively stable arene oxide, probably the 4,5-oxide, which is resistant to the action of epoxide hydrolase.
To date, the metabolic formation of bay-region diol epoxides is the only known pathway by which polycyclic aromatic hydrocarbons are activated to ultimate carcinogens (for recent reviews cfi Refs. 1 and 2). For benzo(a)pyrene (31, benz(a)anthracene (41, and chrysene (51, the (+)-~~,S)-diol-(S~~-epoxides((+)-diol epoxide-2) have been shown to be the isomers * The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18  responsible for the tumorigenic activity. In the case of BP,' alternate mechanisms of metabolic activation which involve the 6-position have been proposed (6,7). Although substitution of BP at the 6-position generally reduces tumorigenic activity on mouse skin (8-ll), the actual basis for the reduction in activity is presently unknown. Comparison of results obtained with BP and with 6-fluorobenzo(a)pyrene provides an interesting case. In terms of bay-region activation via the diol epoxide pathway, both hydrocarbons are metabolized by hepatic mic~somal c~ochrome P-450 at about the same rate, and nearly twice as much 7,8-dihydrodiol, as a percentage of total metabolites, is formed from FBP (11). Although this latter result is suggestive that fluorination at the 6-position of FBP might enhance tumorigenic activity via the bay-region pathway, metabolic activation of the 7,8-&hydrodiol from FBP produced a far weaker mutagenic response toward Chinese hamster V79 cells than did the 7,8-dihy~odiol from BP (11). The present study evaluates two possible causes for this decrease in mutagenic response from the flu or in at^ 7,8-dihydrodiol: that the fluorinated 7,8-diol-9,10-epoxides are metabolically formed at a lower rate and that the fluorinated 7,8-diol-9,10-epoxides have lower inherent mutagenic activity. Both of these factors could contribute to altered tumorigenicity of FBP relative to BP.

General
HPLC analyses were performed on a Spectra-Physics Model 3500B liquid chromatograph equipped with a Schoeffel Model 770 variable wavelength detector and a HP 3390A integrator. Radioactivity was measured in Aquasol with an Intertechnique SL 4200 liquid scintillation counter. DuPont Zorbax SIL and ODS columns were used as indicated. UV spectra were recorded with a Hewlett-Packard Model 8450A UV/VIS spectrophotometer either in conventional cuvettes in the static mode or in a flow cell for HPLC effluents. NMR spectra were recorded with a JEOL FX-100 spectrometer (100 MHz) or with a Nicotet spectrometer (500 MHz). Chemical ionization mass spectra were measured with a Finnegan Model 1015D combined gas chromatograph-mass spectrometer. Circular dichroism spectra were measured with a JASCO J500A circular dichroism spectrophotometer.
The total quinone product from either of the above reactions was dissolved in 150 ml of isopropyl alcohol with sonication, 3 mg of sodium borohydride was added, and the mixture was stirred at room temperature. Reduction to the desired racemic dihydrodiol was complete within 10 min for the 4,5-quinone hut required 16 h for the 7,8quinone. After evaporation of the isopropyl alcohol, the residue was dissolved in ethyl acetate and subjected to standard workup. Although the racemic FBP 4,5-dihydrodiol was sufficiently pure for further studies, the racemic FBP 7,~-dihydrodiol was purified by chromatography on the above Radial Pak column eluted with 50% ethyl acetate in dichloromethane at a flow rate of 8.8 ml/min; retention time 1.6 min. For both racemic ~hydrodiols, the overall yield for the two steps exceeded 70%. They were identical with the corresponding metabolically produced compounds by NMR spectra (11) and retention time on HPLC. Neither technique gave evidence that any cis dihydro~ol had formed in the reduction step.

Diastereomeric Bis-esters of ~i h y d r o d~o l s and T e~r a h y d r~i o l s
The 4,5-dihydrodiol of FBP is quite stable and readily forms a diastereomeric pair of bis-esters with the acid chloride of MAA (1 mg of dihydrodiol and 40 mg of acid chloride in 100 pi of pyridine at room temperature for 12 h). In contrast, related bis-esters of the metabolically formed FBP 7,8-and 9,lO-dihydrodiols were not readily isolated, partly due to their instability. Thus, the metabolically formed 7,8-and 9,lO-dihydrodiol as well as the racemic 7,8-dihydrodiol of FBP were first reduced (1 atm of Hz, Pt) in ethyl acetate to saturate their nonaromatic double bonds. The resulting H4-9,10-diol from the metabolite as well as a semisynthetic sample enriched ( 2 1 ) in the (S,S)-enantiomer' were converted to bis-MAA esters as described for the 4,5-dihydrodiol except that a trace of p-dimethylaminopyridine was added to accelerate reaction. Better chromatographic resolution dictated the use of bis-MTPA esters for separation of the diastereomeric esters from the H4-7,8-diol. The bis-MTPA esters were readily formed (12 h in dry pyridine) and were stable. Separation of all the diastereomers will be described under "Results."

Tetraofs from F E P 7,8-Dwl-9,lo-epox~des
Metabolically formed (-)-FBP (7R,8R)-dihydrodiol (>98% enantiomerically pure) isolated in the present study was epoxidized to its diastereomeric pair of 7,8-diol-9,lO-epoxides (isomer-l in which the benzylic 7-hydroxyl and epoxide oxygen are cis and isomer-2 in which these groups are trans) as described separately.2 The pure diol epoxides were used in the mutagenesis experiments described here and were hydrolyzed to tetraols by both cis and trans hydration of the epoxides at the benzylic 10-position. Relative stereochemistry of the tetraols was deduced from the NMR spectra of their tetraacetates?

Enzymes
Glucose-6-phosphate dehydrogenase (Type XII, sulfate-free) was purchased from Sigma. Liver microsomes were prepared from immature (50-60 g) male rats of the Long-Evans strain after prior treatment with 3-methylcholanthrene as described previously (17). The cytochrome P-450 content was 1.61 nmol/mg of protein as determined spectrophotometrically (18). The highly purified monooxygenase system was reconstituted with lipid, cytochrome P-45Oc (19), and NADPH-cytochrome c reductase (20). Incubations with the purified and reconstituted system were performed in the presence and absence of homogeneous microsomal epoxide hydrolase (EC 3.3.2.3) (21). For incubations with the highly purified and reconstituted system containing cytochrome P-45Oc, microsomes were replaced by 0.1-0.4 nmol of cytochrome P-45Oc, 1500-3000 units of NADPH-cytochrome c reductase, and 50 pg of dilauroylphosphatidylcholine. These incubations were performed either in the absence or in the presence of 32 pg of purified epoxide hydrolase. After incubations at 37 "C for 10 min, the reaction was stopped by addition of 2 ml of acetone and 4 ml of ethyl acetate. Metabolites and residual substrate were extracted into the organic phase which was then separated, dried (anhydrous NaZS04), and evaporated with a stream of nitrogen.

M u t~e n e s~s Assays with Bacteria and Mammalian Cells
Strains TA 98 and TA 100 of Salmonella typhimurium (22) were obtained from Dr. B. Ames, University of California (Berkeley) and cultured for 8 h as described (23). Epoxides were added in 15 pl of anhydrous dimethyl sulfoxide to 2 X lo8 bacteria suspended in 0.5 ml of phosphate-buffered saline (5 mM potassium phosphate, 150 mM sodium chloride, pH 7.0). Assay mixtures were incubated for 5 min at 37 "C before plating on minimal agar medium. Mutations from histidine dependence to histidine independence were assessed 48 h after plating by counting the macroscopic colonies of bacteria on the Petri dishes. Mutation frequencies obtained from at least three replicates in each experiment were analyzed as previously described (24).
The Chinese hamster cell line V79-6 was obtained from by Dr. E. H. Y. Chu, University of Michigan (Ann Arbor). Resistance to the lethal effects of the purine analog 8-azaguanine was used as the mutagenic marker. Procedures for culturing the cells, assessing toxicity, and inducing 8-azaguanine-resistant variants were adapted from Chu (25) and the conditions used were as described previously (23). Epoxides were added in 20 p1 of anhydrous dimethyl sulfoxide to cells which were growing in 5 mi of cuiture medium. In each of at least 2 separate experiments for each compound, 4 and 16 replicate dishes were used to assess cell survival and 8-azaguanine resistance, respectively. The spontaneous mutation frequency for 3 experiments averaged 0.13 * 0.08 (S.E.) 8-aza~anine-resis~nt colonies/lO' surviving cells and never exceeded a value of 0.26. The absolute plating efficiencies of the cells averaged 90 rf-. 4 (S.E.) per cent for the 3 experiments and was never less than 82%.

RESULTS
Metabolism of FBP-The FBP 7,s-dihydrodiol required for the synthesis of the bay-region FBP 7,s-diol-9,10-epoxides' was obtained by preparative scale incubation of FBP with hepatic microsomes from 3-methycholanthrene-treated rats. In order to optimize the yield of the dihydrodiol, both the total metabolism of FBP and the amount of FBP 7,s-dihydrodiol formed were examined as a function of protein concentration (Fig. 1). As the protein concentration was increased from 0.9 mg/ml to 1.4 mg/ml, the percentage conversion of FBP went from 76% (6.1 pmol) to 92% (7.4 pmol); however, the yield of FBP 7,s-dihydrodiol increased by more than 2fold from 0.5 pmol to 1.23 pmol per 50-ml incubation containing 8 pmol of FBP (8-17% of total metabolites). Further increase in protein concentration to 1.8 mg/ml did not significantly alter the yield of the 7,8-dihydrodiol. At an even higher protein concentration of 2.6 mg/ml, the yield of FBP 7,sdihydrodiol dropped to 0.6 pmol per incubation (8% of total metabolites). At protein concentrations below 1.4 mg/ml, the lower percentage of 7,s-dihydrodiol formed together with a concomitant increase in the phenols formed (data for phenols not shown) appears to be caused by an insufficient amount of epoxide hydrolase relative to cytochromes P-450. At protein concentrations above 1.8 mg/ml, the lower yield of the 7,sdihydrodiol presumably reflects secondary metabolism of the dihydrodiol by the monooxygenase systems present. Interestingly, when the incubation was carried out in a 250-ml flask instead of a 500-ml flask, the conversion of FBP dropped from 76% (6.1 pmol) to 46% (3.7 pmol) at 0.9 mg/ml microsomal protein with no change in the yield of FBP 7,s-dihydrodiol. An insufficient supply of oxygen in the incubation medium in the 250-mi flask appears to be the cause for reduced conversion. Even in the 500-ml flask, the amount of oxygen must be somewhat limited since aeration of the incubation medium periodically during the 20-min incubation period increased the overall conversion of FBP by about 15% (Fig.  1). These experiments led to the conditions of incubation which gave optimum yield of FBP 7,s-dihydrodiol. Thus, 28 mg (93 pmol) of FBP 7,s-dihydrodiol was obtained by incubation of 800 pmol of FBP in 100 Erlenmeyer flasks (500-ml capacity) with 7 g of hepatic microsomal protein from 3methylcholanthrene-treated rats for 20 min. In addition, 12 mg of the 4,5-dihydrodiol and 15 mg of the 9,lO-dihydrodiol were obtained. Enantiomeric Purity and Absolute Configuration of the 4,5-, 7,8-, and 9,lO-Dihydrodiols Metabolically Formed from FBP-Assignment of enantiomeric purity to the dihydrodiols was based on the HPLC separation of derived diastereomeric bis-esters with optically active acids (Table I). Since none of the three dihydrodiols is presently available in racemic form by synthesis, the 4,5-and 7,s-dihydrodiols were prepared in racemic form by (i) oxidation to the achiral quinones with 2,3-dichloro-5,6-dicyano-1,4-benzoquinone followed by (ii) reduction of the quinones back to the racemic trans dihydrodiols (cf. Refs. 26 and 27). The cyclic sequence was effected with excellent yields a t both steps. ~i~s t e r e o m e r separation as the bis-MAA ester was achieved directly on the racemic 4,5dihydrodiol with an 80% valley between the peaks. For the metabolically formed dihydrodiol, 98% chromatographed as the diastereomer (three recycles). No attempt was made to establish that the 2% which chromatographed as the (S,S)diastereomer peak was actually this compound. For reasons of stability, the racemic and metabolically formed 7,8-dihydrodiol was first reduced to the H4-7,8-diol before conversion to bis-MTPA esters; 80% valley for racemic. In this case, the minor peak (<2%) which corresponded to the (S,S)-diastereomer was found to have the expected UV spectrum. The 'Chromato~aphed as the bis-MAA esters of the me~bolically formed FBP 4,5-dihydrodiol on a DuPont Zorbax SIL column (0.62 X 25 cm) eluted with 8% ether in cyclohexane at a flow rate of 6.6 ml/min. 'Chromatographed as the bis-MTPA esters of the H4-7,8-diol obtained by reduction of the metabolically formed FBP 7,8-dihydrodiol; DuPont Zorbax ODS column (0.46 X 25 cm) eluted with a wateracetonitrile gradient from 90% acetonitrile in water to 100% acetonitrile (l%/min) at a flow rate of 2.0 ml/min. ~Chroma~graphed as the bis-MAA esters of the H4-9,10-diol obtained by reduction of the metabolically formed FBP 9,10-dihydrodiol; DuPont Golden SIL column (0.62 X 10 cm, 3 p ) eluted with 10% ether in cyclohexane at, a flow rate of 2.2 ml/min.  (-) and of (B) FBP Q,lO-dihydrodiol(-).
As previously noted (ll), circular dichroism spectra of closely related dihydrodiols may be used to infer absolute configuration prouided their preferred c o~f o r~a~~~ are identical. Evidence based on HPLC retention times suggests that BP 4,5-dihydrodiol is mainly pseudodiequatorial while FBP 4,5-dihydrodiol is mainly pseudodiaxial (11). Although the NMR spectrum of FBP 4,5-dihydrodiol confirms that its hydroxyl groups prefer the pseudodiaxial conformation ( U ) , magnetic equivalence of the hydrogens at C-4 and C-5 in BP 4,5-dihydrodiol precludes the use of NMR to assign its conformation. However, the 500-MHz NMR spectra (CDC13) of the diacetates of BP 4,5-dihydrodiol and FBP 4,5-dihydrodiol revealed coupling constants of 4.0 Hz and 2.0 Hz, respectively, between the hydrogens at C4 andCs, indicating that the acetoxy groups prefer the pseudo~axial confo~ation in both esters. The marked similarity of the CD spectra (Fig. 2 4 ) of the diacetates of the (-)-FBP 4,5-dihydrodiol metabolite and of (+)-BP (4R,5R)-dihydrodiol (28) indicates that (4R,5R)absolute confi~ration is common to both dihydrodiols despite the fact that they have opposite signs for their values of [aID (tetrahydrofuran). Although the CD spectra (methanol) and NMR spectra (chloroform) were run in different solvents, this should not significantly affect the conformations of the dihydrodiols as their diacetates. Because the hydroxyl groups of both BP and FBP 9,10-dihydrodiol form parts of bayregions, steric hindrance causes them to prefer the pseudodiaxial conformation (11,29). Thus, their CD spectra can be directly compared. The marked similarity of the CD spectra ( Fig. 2B) of (-)-BP (SR,lOR)-dihydrodiol (30) and the (-1- An alternate method of assigning absolute configuration to trans dihydro-and tetrahydrodiols of polycylic aromatic hydrocarbons is based on the NMR spectra (CsD,) of their bis-MAA esters-For the (SSf-diastereomers, the hydrogens of the -OCH2COc groups are magneticaliy nonequivalent whereas these hydrogens are magnetically equivalent or very  (Table I) each of these groups appears as an AB-quartet centered at 6 3.50 and 3.62 with Jgem = 16 Hz. A similar result pertains to the enantiomers of the FBP H4-9,lO-diol as its bis-MAA esters. Thus, both the CD and NMR methods lead to the same assignments as designated in Table   I.
Metabolism of (-)-FBP (7R,8R)-DihydrodioL"he semisynthetic FBP 7,8-dioI-9,lO-epoxide diastereomers-1 and -2 were found to hydrolyze completely to tetraols (Fig. 3) upon incubation with microsomes in the absence of NADPH, work-up, and chromatographic analysis. This is consistent with the observed spectrophotmetric half-life (0.10 M phosphate, pH 7.4,37 "C) of 88 s for diol epoxide-2 under the conditions of incubation. Notably, under the solvolysis conditions used to study the chemical hydrolysis* of diol epoxide-2 (pH 2 7, 1 mM buffer containing 0.1 M NaCIOl and 10% dioxane, 25 "c), -40% of the products consists of the 9-keto 7,8-diol under these spontaneous hydrolysis conditions. Although this keto diol is not detected chromatographically, it is readily identifiable after reduction with sodium borohydride to a diastereomeric pair of 7,8,9-triols. Application of this technique to FBP diol epoxide-2 which had been subjected to the incubation conditions failed to produce detectable triol. The absence of detectable keto diol which forms under the conditions of spontaneous hydrolysis is probably due to the fact that about half of the diol epoxide hydrolysis which occurs in the incu- Metabolism of (-)-FBP (7R,8R)-dihydrodwl by liver microsomes from 3-methykholanthrene-treated rats and by a highly purified and reconstituted system containing cytochrome P-45Oc with or without epoxide hydrohe Experimental conditions were as described under "Materials and Methods." The substrate concentration was 100 nmol/2.0-ml incubation. Data are expressed as the percentage of each metabolite compared with total metabolites eluting in the defined peaks. ' Per cent metabolism denotes the percentage conversion of substrate; i.e. total radioactivity above blank which emerges from the column before the substrate. Total metabolism does not take into account traces of unextracted metabolites, highly polar and nonextracted metabolites, or covalently bound metabolites which remain in the aqueous phase after extraction. Recovery (the percentage of the total radioactivity due to metabolism emerging from the column before the substrate in defined metabolite peaks as compared with total radioactivity due to metabolism) was 64-77% for the above incubation conditions. Rate of metabolism given in parentheses is expressed as nanomoles of extracted metabolites/nmol of cytochrome P-450c/min. a Within each of the 3 pairs of epoxides, the mutagenic activity of the unsubstituted BP epoxide was assigned a relative activity of 100. The activities of the 6-fluor0 analogs are expressed as a percentage of the activity observed with the unsubstituted epoxide.
Reversion of histidine-dependent growth to histidine-independent growth in strains TA 98 and TA 100 of S. typhimurium was assessed as previously described (24) utilizing 4 concentrations of each epoxide.
Induction of 8-azaguanine resistance in cultured Chinese hamster V79 cells was assessed as previously described (25), utilizing from 3-4 concentrations of each epoxide.
The enantiomeric composition of FBP H4-9,10-epoxide2 is 68% (SS,lOR)-enantiomer and 32% (SR,lOS)-enantiomer. For purposes of comparison, the ratio of mutagenic activity of the (+)-and (-)enantiomers of BP HI-9,lO-epoxide did not differ by more than a factor of 1.3 in either strain of S. typhimurium. radioactive (-)-FBP (7R,8R)-dihydrodiol with liver microsomes from 3-methylcholanthrene-treated rats and with the highly purified monooxygenase system reconstituted with cytochrome P-45Oc are given in Table 11. A typical metabolite profile and the HPLC conditions are shown in Fig. 4. Since the tetraols which arise from diol epoxide-1 were minor metabolites (<7%), chromatographic conditions were selected to optimize the separation of other metabolites. Tetraols, identified by their UV spectra and retention times, ranged from 38% to 55% of the total metabolites. The next most significant group of metabolites consisted of three phenolic dihydrodiols. All three metabolites have similar UV absorption bands between 200 nm and 300 nm, but have significant differences in their spectra between 300 nm and 400 nm (Fig. 5A). The phenolic nature of these metabolites was apparent from the bathochromic shift of their absorption bands in alkaline SOlutions (Fig. 5A, inset) and by an increase of 16 mass units over that of the dihydrodiol as apparent from their molecular The metabolite that elutes just after phenolic dihydrodiols-1 and -2 has a UV spectrum similar to that of chrysene 1,2-dihydrodiol (33) (Fig. 5B) indicating that it is probably an arene oxide at the 4,5-position of FBP 7,8-dihydrodiol. From experiments with the reconstituted system in the presence of epoxide hydrolase, it is clear that the arene oxide is highly resistant to the action of this enzyme. The arene oxide is labile and readily isomerizes in the presence of a trace amount of acid to form the phenolic dihydrodiol-3. Although the total amount of the arene oxide and resultant phenolic dihydrodiol remained constant for a given incubation, their ratio varied depending upon the particular ODS column utilized for the analysis. Structures of the various metabolites are shown in Fig. 6.

DISCUSSION
As is the case for the metabolism of BP, the combined action of hepatic microsomal cytochrome P-450 and epoxide hydrolase converts FBP to (R,R)-dihydrodiols at the 4,5-, 7,8-, and 9,10-positions with very high enantiomeric purity. Based on a steric model for the catalytic binding site of cytochrome P-45Oc (34), the only known cytochrome P-450 isozyme which has a very high turnover number for the oxidation of polycyclic aromatic hydrocarbons, this result was to be expected since the two hydrocarbons are isosteric. The site model also accomodates the fact that (-)-BP (7R,8R)dihydrodiol is converted mainly to the 7,8-diol-9,10-epoxide-2 diastereomer (81-86% of total diol epoxides) relative to the diol epoxide-1 diastereomer. Since the reverse is true (diol epoxide-1 favored 2 0 1 over diol epoxide-2) for (+)-BP (7S,8S)-dihydrodiol, cytochrome P-45Oc shows a marked preference for a specific stereoheterotrophic face of BP 7,8-dihydrodiol and ignores the absolute configuration of the tram diol group. Consistent with the model, cytochrome P -4 5 0~ favors formation of the diol epoxide-2 (83-89% of total diol epoxides) relative to the diol epoxide-1 diastereomer of (-)-FBP (( 7R,8R)-dihydrodiol. The parameter of altered substrate conformation has been introduced through study of the metabolism of the fluorinated dihydrodiol. Unlike B P 7,8-dihy-drodiol, the FBP 7,8-dihydrodiol prefers the pseudodiaxial rather than the pseudodiequatorial conformation for its hydroxyl groups due to adverse electrostatic interaction between the proximate fluorine and hydroxyl substituents when in the pseudodiequatorial conformation. Thus, at least in the cases of the metabolism of the 7,8-dihydrodiol from BP and FBP, the predicted facial preference of cytochrome P-45Oc at the 9,10-position is practically unaffected by either the absolute configuration (compare (+)-and (-)-7&dihydrodiols of B P and (-)-dihydrodiol of FBP) or preferred conformation (compare (-)-BP 7,8-dihydrodiol with (-)-FBP 7,8-dihydrodiol).
We had previously observed that two other pseudodiaxial, benzo-ring dihydrodiols, B P 9,lO-dihydrodiol (35) and benzo(e)pyrene 9,10-dihydrodiol (36) formed at best only trace amounts of benzo-ring diol epoxides on metabolism by cytochrome P-45Oc or liver microsomes from 3-methylcholanthrene-treated rats despite the obvious chemical preference for oxidation at the nonaromatic double bond. Hence, we had proposed that the increased bulk and/or polarity of the pseudodiaxial hydroxyl groups inhibited metabolism at the adjacent double bond (35,36). Although several conflicting reports (37)(38)(39) have attempted to address this question, a definitive conclusion has not been forthcoming (27). The present study represents the first example where the metabolism of the same regioisomer of a dihydrodiol in either the pseudodiaxial or -diequatorial conformation, depending on the presence or absence of an adjacent perifluorine substituent (40), has been studied. Three features are salient. First, neither the overall rate of metabolism nor the amount of 9,10-dihydrodiol as a per cent of total metabolites is changed by introduction of the 6-fluor0 substituent into B P when metabolized by liver microsomes from 3-methylcholanthrene-treated rats (11). Thus, fluorination at the 6-position does not affect the regiospecificity of cytochrome P-45Oc for the 9,10-double bond when the two hydrocarbons are compared. Second, the rates at which cytochrome P-45Oc oxidizes FBP 7,8-dihydrodiol (10 nmol/nmol of cytochrome P-450c/min) and B P 7,a-dihydrodiol (cf. Ref. 15 and redetermined in the present study as 8 nmol/nmol of cytochrome P-450c/min) are essentially identical. Third, bay-region diol epoxides account for about 95% of the metabolites from (-)-BP 7,8-dihydrodiol but only 38-43% of the metabolites from (-)-FBP 7,8-dihydrodiol with cytochrome P-45Oc. Regardless of the mechanism, we interpret these results as clear evidence that pseudodiaxial hydroxyl groups inhibit metabolism at an adjacent benzo-ring, double-bond relative to pseudodiequatorial hydroxyl groups, in contrast to the conclusions of other workers (38,39).
A particularly interesting aspect of the present study has been the tentative identification of a K-region, arene 4,soxide as a relatively stable metabolite of FBP 7,8-dihydrodiol. Because the metabolite is a relatively minor product with microsomes and because it readily isomerizes to phenolic dihydrodiol-3, attempts were not made to isolate a sufficient quantity for an NMR spectrum. The similarity of its UV spectrum to that of chrysene 1,2-dihydrodiol rather than benz(a)anthracene 8,9-dihydrodiol along with its relative stability argue for a K-region arene oxide at the 4,5-position of the FBP 7,8-dihydrodiol. The site model for cytochrome P-450c predicts that the FBP 7,8-dihydrodiol as well as BP are attacked from the same face during epoxide formation at the 4,5-position. The main diastereomer produced would thus have the epoxide group (4S,5R-absolute configuration) trans to the benzylic 7-hydroxyl group with FBP 7,8-dihydrodiol as the substrate. This particular stereoisomer would be predicted to bind poorly to the catalytic site of epoxide hydrolase (41) since the diol group would reside in the hydrophobic pocket of the enzyme. This argument perhaps accounts for the fact that an amount of epoxide hydrolase (16 pg/ml) sufficient to hydrate completely 3 times the amount of B P 4,5-oxide formed in the reconstituted system (42) failed to metabolize a detectable amount of the FBP 7,8-diol-4,5-oxide. The low solvolytic reactivity and recalcitrance of the metabolite toward epoxide hydrolase is reminiscent of a recently isolated arene oxide metabolite of P-naphthoflavone (43)(44)(45).
(-)-FBP (7R,8R)-dihydrodiol produces a far weaker mutagenic response than does (-)-BP (7R,8R)-dihydrodiol when metabolically activated by liver microsomes from 3-methylcholanthrene-treated rats in the presence of Chinese hamster V79 cells (11). Results of the present study show that the markedly decreased mutagenic response is not explicable in terms of the decreased conversion of FBP 7,8-dihydrodiol to bay-region diol epoxides. For this reason, the diastereomeric 7,8-diol-9,10-epoxides of (-)-FBP (7R,8R)-dihydrodiol were compared to their B P counterparts for inherent mutagenic activity toward bacterial and mammalian cells. A several fold decrease in mutagenic activity was observed for the fluorinated diol epoxides relative to the unfluorinated compounds in the three test systems. Although changes in gross chemical reactivity due to fluorine substitution might be responsible for the decreased mutagenicity, this does not seem to be the case. At neutral to alkaline pH in the absence of buffer catalysis, the FBP 7,8-diol-9,10-epoxide-2 diastereomer undergoes spontaneous hydrolysis to tetraols at about 3 times the rate of its highly tumorigenic (+)-BP (7R,8S)-diol-(SS,lOR)-epoxide counterpart.' The small increase in rate for the fluorinated molecule has been attributed to competing inductive and stereoelectronic factors. Notably, in the phosphate-buffered saline medium at 37 "C used for the bacterial mutagenesis assays, B P diol epoxide-2 reacts approximately 1.5 times faster than its fluorinated analog which has a halflife of -2 min. This reversal in reactivity results from differential susceptibility of the two diol epoxides to general acidcatalyzed hydrolysis by the 5 mM phosphate buffer. Thus, the solvolytic lifetime of the fluorinated FBP diol epoxide-2 is actually longer than that of B P diol epoxide-2 under conditions of the bacterial mutagenesis assay. Since the FBP diol epoxide-l diastereomer is 240-fold less reactive (much longer lifetimes) than its B P counterpart toward spontaneous solvolysis, there was no need to check for differential susceptibility to phosphate catalysis with the fluorinated and unfluorinated diol epoxide-l diastereomers. In conclusion, the severalfold decrease in mutagenic activity on fluorination of the BP diol epoxide-l and -2 diastereomers is not due to decreased solvolytic lifetime since the fluorinated compounds have comparable to greatly increased solvolytic half-lifes. In view of the above findings, we feel that future comparisons of relative mutagenic activity for reactive metabolites should attempt to take into consideration such differences in solvolytic lifetime.
Comparisons of mutagenic activity between the BP and FBP 7,8-diol-9,10-epoxide-2 isomers were made for compounds with the same absolute configurations, (7R,8S)-diol-(9S,lOR)-epoxide. This point is important since marked differences in mutagenic activity have been noted between enantiomers, especially toward Chinese hamster V79 cells (46)(47)(48)(49). In contrast, bay-region tetrahydro epoxides show little difference in mutagenic activity between enantiomers (cf .  Table 111 and Refs. 47 and 48). The data in Table 111 indicate that the presence of a 6-fluor0 substituent on B P H,-9,10epoxide has only a minor retarding effect (0-25%) on mutagenic activity toward the bacterial and mammalian cells. In the phosphate-buffered saline medium used with the bacterial cells, B P H4-9,lO-epoxide has a solvolytic half-life of 37 s and FBP H4-9,10-epoxide has a half-life of 420 s. The competing effects, 10-fold increased half-life versus decreased chemical reactivity for the FBP H4-9,10-epoxide, appear to cause comparable mutagenic activity for the fluorinated and unfluorinated 9,10-epoxides. At present, our best explanation for the 8147% decrease in mutagenic activity of the fluorinated diol epoxides toward the mammalian cells is that it is a consequence of the markedly enhanced preference of these diol epoxides for the conformation in which their hydroxyl groups are pseudodiaxial.* The decreased tumorigenic activity of FBP relative to BP on mouse skin cannot be explained as a direct consequence of differences in metabolism. In terms of the bay-region diol epoxide pathway, the differences tend to balance each other out. Although twice as much 7,8-dihydrodiol is formed from FBP, it is only converted half as well to bay-region diol epoxides. Thus, both hydrocarbons form about the same amount of diol epoxide, of which the (7R,SS)-diol-(SS,lOR)epoxide stereoisomer greatly predominates in each case. Solvolytic reactivity of the fluorinated and unfluorinated diol epoxide-2 stereoisomers is comparable but preferred conformation is not. The hydroxyl groups in the fluoro diol epoxide have been found to prefer the pseudodiaxial conformation. Whether or not this marked change in conformation affects tumorigenic response is under test.