THE JOURNAL OF BIOLOGICAL CHEMISTRY

Printed in U.S.A. Vol. 258. No. 9, Issue of May 10, pp. 5649-5659, 1983

Metabolism of a-Naphthoflavone and 8-Naphthoflavone by Rat Liver Microsomes and Highly Purified Reconstituted Cytochrome P-450 Systems*

(Received for publication, November 15, 1982)

Kamlesh P. VyasSg, Tetsuichi ShibataST, Robert J. HighetS, Herman J. YehS, Paul E. Thomasll, Dene E. Ryan((, Wayne LevinJI, and Donald M. Jerina+** From the $National Institute of Arthritis, Diabetes, and Digestive and Kidney Diseases and the National Institute of Heart, Lung, and Blood, National Institutes of Health, Bethesda, Maryland 20205 and the IlDepartment of Biochemistry and Drug Metabolism, Hoffmann-La Roche Inc., Nutley, New Jersey 07110

Metabolism of 8-naphthoflavone (BNF) and a-naph- thoflavone (ANF) by liver microsomes from control and treated rats, as well as by a purified cytochrome P-450 system, has been investigated. Liver microsomes from control and phenobarbital-treated rats metabolized BNF mainly to 8-hydroxy-BNF, whereas the tmns-7,8- dihydrodiol was the major metabolite formed by liver microsomes from 3-methylcholanthrene- or BNF- treated rats and by a purified system containing cyto- chrome P-45Oc and epoxide hydrolase. Other metabo- lites were a tmm-5,6-dihydrodiol and 5-hydroxy-BNF. Rates of metabolism (nanomoles of metabolites/nmol of cytochrome P-450/min) indicated that both 3-meth- ylcholanthrene and BNF treatment of rats induced me- tabolism 2-2.5-fold compared to untreated rats, whereas phenobarbital treatment of rats decreased the rate by 50%. All three treatments caused a 34-41% decrease in the rate of metabolism of ANF compared to control. The 5,6-oxide was the major metabolite and the 5,6-dihydrodiol a minor metabolite with all the enzyme systems. Homogeneous epoxide hydrolase (EC 3.3.2.3) metabolizes this relatively stable arene oxide at a very slow rate (1 nmol/min/mg of protein). Other major metabolites were 6-hydroxy-ANF and another dihydrodiol. Although it had been concluded from ear- lier studies that this was the 9,10-dihydrodiol, our re- sults indicate it is actually the 7,8-dihydrodiol. When the purified system was reconstituted with cytochrome P-450b, detectable metabolites were not observed with either ANF or BNF as substrates, a result consistent with the fact that anti-P-450b failed to inhibit micro- somal metabolism of these substrates. By comparison anti-P-450c markedly inhibited the metabolism of BNF but not ANF by liver microsomes from 3-methylchol- anthrene-treated rats.

ANF’ and BNF, two synthetic derivatives of a large class of

* 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 U.S.C. Section 1734 solely to indicate this fact.

5 Present address, Department of Drug Metabolism, Merck Sharp and Dohme Research Laboratories, West Point, PA 19486.

7 Present address, School of Medicine, Keio University, 35 Shin- anomachi, Shinjuku-Ku, Tokyo 160, Japan.

* * T o whom correspondence should be addressed at The National Institutes of Health, National Institute of Arthritis, Diabetes, and Digestive and Kidney Diseases, Building 4, Room 216, Bethesda, MD 20205.

The abbreviations used are: ANF, a-naphthoflavone (7,8-benzo-

” -~ ~ ~~~~~ ~~

naturally occurring flavonoid compounds, exert pronounced and differential effects on the microsomal cytochrome P-450- dependent monooxygenase system involved in the metabolic activation and detoxification of certain procarcinogens. BNF, but not ANF, is a potent inducer of a cytochrome P-450 isozyme that has high activity for the metabolism of polycyclic aromatic hydrocarbons to ultimate carcinogenic metabolites (1). ANF has been shown to be a potent, selective in vitro inhibitor of cytochrome P-45Oc (1) but is an in uitro activator of several other isozymes (1, 2). Flavone has recently been shown to be an activator of zoxazolamine metabolism in oivo in neonatal rats (3) . The discovery of the inhibitory and inducing properties of naphthoflavones on the cytochrome P- 450-dependent monooxygenase system has led to extensive examination of these flavonoids as modifiers of chemical car- cinogenesis (4,5). Their role in the inhibition of tumorigenesis caused by certain polycyclic aromatic hydrocarbons may re- sult from their multiple and differential effects on the cyto- chrome P-450-dependent monooxygenase system.

Despite the widespread use of ANF and BNF as modulators of metabolism and tumorigenicity, practically nothing has been reported about their metabolism until very recently. Initially, Stegeman and Woodin (6) reported that hepatic microsomes from a marine fish (scup, Stenotomus versicolor) catalyzed the formation of a dihydrodiol from ANF along with two minor metabolites. Although the structure of the dihy- drodiol was not determined, its mass spectral fragmentation pattern suggested that the dihydrodiol group was not on the phenyl substituent. Thakker et al. ( 7 ) , in studying the in uitro effects of ANF on the metabolism and metabolic activation of benzo[e]pyrene 9,lO-dihydrodiol to mutagens, noted that he- patic microsomes from several mammalian species catalyzed the formation of dihydrodiols from ANF. Nesnow et al. (8) first reported the formation of a stable 5,6-arene oxide of ANF. Subsequent reports (9, 10) provided additional confir- mation of this remarkably stable arene oxide metabolite which appears to isomerize to 6-hydroxy ANF (10, 11). With liver microsomes from phenobarbital- and BNF-treated rats, Nes- now and Bergman (10) reported the formation of a 5,6-dihy- drodiol from ANF, particularly after treatment of the rats with BNF. In contrast, Coombs et al. (9) reported the for- mation of a 9,lO-dihydrodiol from ANF with liver microsomes from 3-methylcholanthrene-treated rats and later Coombs claimed (11) that the 5,6-dihydrodiol was only formed when - ~~~~~~~~~~~ . . .

flavone); BNF, P-naphthoflavone (5,6-benaoflavone); ANF 7,8-dihy- drodiol, trans-7,8-dihydroxy-7,8-dihydro ANF (other dihydrodiols are similarly abbreviated); HPLC, high pressure liquid chromatography; 6, parts per million.

5649

by guest on February 28, 2020http://w

ww

.jbc.org/D

ownloaded from

5650 Metabolism of a- and P-Naphthofiauones

the rats were treated with phenobarbital. The present report a t tempts to resolve the above conflicts regarding the nature of the metabolites and the effect of inducing agents on the metabolism of ANF. These results are compared with metab- olism studies of the isomeric flavonoid BNF which, to the best of our knowledge, has not previously been studied. This comparison required the development of a suitable radioactive labeling technique such that accurate quantitation of metab- olites was possible.

EXPERIMENTAL PROCEDURES

Materials-ANF and BNF were purchased from Aldrich and were purified by recrystallization three times from ethanol. Samples of 7- and 9-hydroxy-ANF, synthesized by R. Roth at Midwest Research Institute, were the generous & of S. Nesnow at the USEPA, Re- search Triangle Park, NC. Absorbance spectra were recorded in aqueous methanol with a HP 8450A uv-vis spectrophotometer. Chem- ical ionization (CI) mass spectra were recorded on a Finnigan Model 1015 gas chromatograph/mass spectrograph with the indicated ion- izing gases. NMR spectra were recorded on a Jeol FX-100, a Nicolet NT 360, and a Nicolet NT 500 NMR spectrometer. Chemical shifts are reported in parts per million relative to internal tetra- methylsilane in the solvents indicated.

Synthesis of Tritiated ANF and BNF-A mixture of 525 pl of heptafluorobutyric anhydride and 42 pl of tritiated water (5 Ci/ml) was heated in a Teflon-capped, 5-ml reaction vial at 130 "C for 15 min. The vial was cooled, 15 mg of BNF was added, and the reaction mixture was stirred and heated at 130 k 5 "C for 10 days. After cooling, the contents of the vial were dissolved in 2.0 ml of CHC13. The CHCL layer was first washed with 1 ml of 20% w/v NaOH and then with water (3 X 1.5 ml), dried over anhydrous Na2SO4, and concentrated. The BNF was purified by preparative tlc on silica gel G (Analtech, 20 X 20 cm, 250 pm) eluted with 20% ethyl acetate in n- hexane to provide material with a specific activity of 47.8 mCi/mmol. ANF was similarly tritiated except that the reaction mixture was heated at 110 "C for 7 days (specific activity of 27.8 mCi/mmol). Extinction coefficients of 24,300 at 273 nm (methanol) for BNF and 31,200 at 280 nm (methanol) for ANF were used in the determination of specific activities. The radiochemical purity of both flavones was found to be >99% when analyzed by HPLC as described for incubated samples.

Enzyme Preparations-Liver microsomes were prepared from con- trol, phenobarbital-treated (75 mg/kg/day for 3 days), 3-methylchol- anthrene-treated (25 mg/kg/day for 4 days), and BNF-treated (50 mg/kg/day for 4 days) immature, male rats (50-60 g) of the Long- Evans strain as described (12). Microsomal protein concentrations were determined by the method of Lowry et al. (13) and cytochrome P-450 contents were measured spectrally (14). Specific contents (na- nomoles cytochrome P-450/mg of protein) of the various liver micro- somal preparations were 0.72 for control, 2.04 for phenobarbital- treated, 1.31 for 3-methylcholanthrene-treated, and 1.63 for BNF- treated rats. Highly purified cytochrome P-45Oc (15), cytochrome P- 450b (15), NADPH-cytochrome C reductase (16), and epoxide hydro- lase (17) were prepared as described. The epoxide hydrolase prepa- ration was obtained from isosafrole-treated rats and had a specific activity of 600 nmol of glycol formed/min/mg of protein at 37 "C with styrene oxide as substrate. One unit of reductase catalyzes the reduc- tion of 1 nmol of cytochrome c/min at 22 "C in 0.3 M potassium phosphate buffer (pH 7.7) containing 0.1 mM EDTA and 0.1 mM NADPH.

Antibody Preparations-Antibodies against purified cytochromes P-450a, P-450b, P-450c, and P-450d were prepared, and the IgG fractions were isolated as described (18-20). Antibodies prepared against purified cytochromes P-45Oc and P-450d inherently cross- react with the heterologous hemoprotein due to the immunochemical relatedness of these proteins (20). These antibodies are termed anti- P-45Oc and anti-P-450d, based on the antigen of immunization. Anti- P-450c was absorbed against partially purified cytochrome P-450d to remove antibodies directed against common antigenic determinants (20). This antibody, which no longer recognizes cytochrome P-450d has been termed anti-P-450c(-d).

Incubations-Unless otherwise indicated, all incubations were per- formed at 37 "C for 10 min. The standard incubation mixture con- tained 200 umol of potassium phosphate buffer (pH 7.4). 6 Nmol of

protein from 3-methylcholanthrene- or BNF-treated rats, and 100 nmol of substrate (dissolved in 40 pl of methanol) in a final volume of 2.0 ml. Incubations with the reconstituted system contained, in addi- tion to NADPH, MgC12, and 200 nmol of substrate (dissolved in 80 pl of methanol), 0.2 nmol of cytochrome P-450b or cytochrome P- 450c, 1500 units of NADPH-cytochrome C reductase, 40 pg of dilau- roylphosphatidylcholine, 200 pmol of potassium phosphate buffer (pH 7.2), and 0-80 pg of homogeneous microsomal epoxide hydrolase in a final volume of 2.0 ml. Incubations were terminated by addition of 2 ml of acetone, and the unreacted substrate and metabolites were extracted in 4 ml of ethyl acetate. After centrifugation, 5 ml of the organic layer was carefully separated, dried over anhydrous Na2S04, concentrated with a nitrogen stream, and stored at -80 "C until the time of analysis.

HPLC Analysis of Metabolites-Analysis of metabolites was car- ried out on a reverse phase HPLC column using a Spectra-Physics Model 3500B HPLC system. Concentrated extracts from the incuba- tions were dissolved in 100 pl of methanol and 25-pl aliquots were injected on a DuPont Zorbax ODS column (6.2 X 250 mm) eluted with a linear gradient (l%/min) of 5(k100% methanol in water at a flow rate of 1.2 ml/min. Effluent was continuously monitored at 254 nm, and 0.36-0.6-ml fractions were collected throughout the metab- olite profile. Radioactivity in each fraction was measured in Aquasol with an Intertechnique SL-4OOO liquid scintillation spectrometer.

Isolation of Metabolites-Large scale incubations were carried out in order to obtain suffkient amounts of metabolites for spectral

o-NAPHTHOFLAVONE IANF)

1.0

- 71

6.8

6 P-NAPHTHOFLAVONE (BNFl

8.2 8.0 7.8 7.6 7.4 PPM

FIG. 1. Nuclear magnetic resonance spectra (360 MHz, CDCls) of ANF (a-naphthoflavone, 7,l-benzoflavone) and of BNF @-naphthoflavone, 5.6-benzoflavone). Assignment of sig- nals in the ANF spectrum are based primarily on the spectrum of Coombs et al. (9) except for the complex signal a t H ~ o which was confi ied by decoupling and calculated spectra. In order to preserve clarity in the spectrum of BNF, the singlet for HS at 7.03 ppm and the broadened doublet (ortho- and meta-coupled) for Hg at 10.08 ppm are not shown. Irridiation at Hs results in 1) the loss of one ortho coupling in H6, 2) the loss of meta coupling in H? to produce a triplet, 3) the loss of para coupling in Hg to produce a sharp pair of doublets, and 4) a sharpening of the doublet for Hs due to long range coupling to

MgC12, 1 pmol of NADPH, 0.2-1.6-mg of microsbmal protein from Hs. Both flavonoids and their metabolites show marked -solvent control or phenobarbital-treated rats and 0.2-0.8 mg of microsomal dependence in their NMR spectra.

"

by guest on February 28, 2020http://w

ww

.jbc.org/D

ownloaded from

Metabolism of a- and P-Naphthoflauones 5651

characterization. In a final volume of 50 ml, the following components were allowed to react for 10 min at 37 "C: 5 mmol of potassium phosphate buffer (pH 7.4), 150 pmol of MgCb, 25 pmol of NADPH, 50-75 mg of microsomal protein from 3-methylcholanthrene- or phenobarbital-treated rats, and 2.5 pmol of unlabeled substrate dis- solved in 1.0 ml of methanol. Metabolites were extracted with 150 ml of ethyl acetate:acetone (2:l). Metabolites were isolated by reverse phase HPLC on a DuPont Zorbax ODS column (9.45 X 250 mm) eluted with a linear gradient (l%/min) of 6O+lOO% methanol in water at a flow rate of 3.2 ml/min. Metabolites were extracted from the column eluent with ethyl acetate (3 x 4 ml). The combined ethyl acetate extract was dried over anhydrous Na2S04, filtered, and con- centrated in vacuum with a rotary evaporator. Due to high instability of the dihydrodiols to acid, all glassware was washed with triethyla- mine before use and all solutions were maintained at 0 "C.

RESULTS

Tritiation of ANF and BNF-Since both flavonoids are relatively stable to acid and were anticipated to have an exchangable hydrogen at position 3, acid-catalyzed exchange with heptafluorobutyric acid (21) seemed a reasonable ap- proach to produce tritiated substrates. Initial studies with deuterioheptafluorobutyric acid indicated that position 3 of ANF was completely exchanged after 7 days at 110 "C based on a 100-MHz NMR spectrum of the product. Similar tritia- tion provided highly labeled ANF, but a substantial decrease in specific activity occurred on purification of the sample by tlc. Similar chromatography of the deuterated ANF resulted in substantial reincorporation of normal hydrogen at position 3. The 360-MHz NMR spectrum (cf. Fig. 1) of the deuterated sample did, however, indicate that deuterium had also been incorporated to a small extent into the phenyl ring and posi- tions 8/9. Rapid tlc of both tritiated flavonoids without letting the plate air dry before extraction provided material of high specific activity. Although the per cent of tritium remaining at position-3 in both flavonoids is unknown, neither substrate suffered detectable exchange in the course of incubation, workup, or analysis of metabolites by HPLC.

Structures of the Isolated BNF Metabolites-Analytical incubations of [3H]BNF with the various microsomal enzyme preparations allowed the identification of as many as nine discrete metabolite fractions (see later). Preparative scale

incubations of unlabeled BNF with liver microsomes from 3- methylcholanthrene- or phenobarbital-treated rats allowed identification of two of the metabolites as dihydrodiols and two as phenols (Fig. 2). An additional phenol of unknown structure (phenol-A) along with four minor unknown metab- olites account for the balance of the oxidation products.

Dihydrodiols-The two major metabolites of BNF formed by liver microsomes from 3-methylcholanthrene-treated rats had ultraviolet spectra (Fig. 3a) which were unaffected by the addition of base. The CI mass spectra (ammonia or isobutane gas) of both gave a molecular ion at m/e 307 (M' + 1) and a fragment ion at m/e 289 (M' + 1 - 18) corresponding to a loss of water. Since the molecular weight of BNF is 272, both spectra indicate dihydrodiol metabolites. For the major 7,8- dihydrodiol, m/e 307 is the base peak whereas the fragment for loss of water at m/e 289 is the base peak for the 5,6- dihydrodiol. The positional assignments of the dihydrodiols are based on their NMR spectra (Table I).

The carbonyl group in BNF has proved to be exceptionally valuable in aiding the interpretation of the NMR spectra of BNF (Fig. 1) and its metabolites (Table 1). The magnetic anisotropy (22) of this carbonyl group causes marked deshield- ing of H5 in BNF such that it appears at 10.08 6. Inspection of the NMR spectra of both dihydrodiols indicated the absence of this signal, the presence of a pair of vinyl hydrogens, and the presence of a pair of methine hydrogens bearing the added hydroxyl groups. Thus the dihydrodiols must be at the 5,6 and 7,8 positions. Assignment of the 5,6-dihydrodiol is based on the marked downfield shift, relative to related dihydrodiols, of H5 which appears at 5.60 6 due to deshielding by the proximate carbonyl oxygen. The unusually small value of J5,6 = 1.9 Hz ( J d i o l ) indicated that this trans-dihydrodiol is pre- ponderantly in the pseudo-diaxial conformation (see "Dis- cussion"). For the 7,8-dihydrodiol, both methine hydrogens appear at their expected chemical shifts (H7, 4.36 S and Ha, 4.72 S ) , but one of the vinyl hydrogens (H5, 8.19 8) is markedly shifted downfield due to its proximity to the carbonyl oxygen. The value of J7,a = 11.0 Hz (Jdi0l) is typical of that for the trans-dihydrodiol which prefers the usual pseudo-diequatorial conformation. Consistent with this conformation is the small

FIG. 2. The cytochrome P-450-cat- alyzed oxidation of BNF to putative arene oxide intermediates. These ar- ene oxides either isomerize to phenols or are hydrated to trans dihydrodiols by epoxide hydrolase (EH). Only relative BNF configurations are implied.

8-HYDROXY

6H 5.6-DIHYDRODIOL

5-HYDROXY

by guest on February 28, 2020http://w

ww

.jbc.org/D

ownloaded from

5652 Metabolism of a- and ,8-Naphthoflauones

A - 7.8-DIHYDRODIOL "" 5.6-DIHYDRODIOL

B 8-HYDROXY - Neutral pH "" Alkaline pH

FIG. 3. Ultraviolet spectra of the metabolites of BNF. A, BNF 5,6- and 7,8-dihydrodiols; B, 8-hydroxy-BNF; C, 5-hydroxy-BNF and D, phenol A re- corded in the water/methanol composi- tion at which they emerged from the HPLC column. rn

Q

WAVELENGTH (nm)

TABLE I NMR spectra of selected metabolites of BNF

- Proton (chemical shifts and coupling constants)

Compound H S H5 H6 H; H8 Hv HI0 2 Aromatic H 3 Aromatic H

5,6-Dihydrodi01".~ 6.90(s) 5.60(d) 4.39(dd) 6.20(dd) 6.65(d) 7,8-Dihydrodioln3' 6.74(s) 8.19(dd) 6.13(dd) 4.36(dt) 4.72(d) 8-Hydroxy"' 6.93(s) 9.55(dd) 7.49(t) 7.04(dd) 5,6-Benzoflavone' 7.03(s) 10.08(d) 7.78(t) 7.64(t) 7.93(d) 8.14(d) 7.65(d) 7.96-8.00(m) 7.54-7.59(m)

8.0-8.2(rn) 7.5-7.7(m) 7.9-8.1 ( r n ) 7.5-7.7(m)

8.67(d) 7.69(d) 8.0-8.16(m) 7.5-7.63(m)

NMR spectrum at 100 MHz in acetone-d6 containing a trace of perdeuteromethanol. Irridiation at Hs in the 5,6-dihydrodiol and at H7 in the 7,8-dihydrodiol confirmed the indicated coupling patterns. The signals for H9 and HIO of the two dihydrodiols are obscured by the signals for the hydrogens of the phenyl substituents.

55.6 = 1.9 HZ; Js.7 = 5.2 HZ; 5 7 . 8 = 10.0 HZ. J5.s = 11.0 Hz; J5.7 = J6.i = 2.0 Hz; 57.8 = 11.0 Hz. NMR spectrum at 360 MHz in acetone-ds. 55,s = J 6 . 7 = 8.0 HZ; J5.7 = 1.0 HZ; 5s.10 = 9.5 HZ.

'NMR spectrum a t 360 MHz in CDCb. See Fig. 1.

value of Js,7 = 2.0 Hz. Phenols-The assignment of 8-hydroxy-BNF as a phenol is

based on a reversible red shift of its ultraviolet spectrum in alkali (Fig. 3B) and its molecular ion at m/e 289 (M' + 1) obtained by CI (methane gas) mass'spectrometry. I t is formed on acid-catalyzed dehydration of the 7,8-dihydrodiol. Experi- ments using the reconstituted system with and without epox- ide hydrolase (see later) indicate that 8-hydroxy-BNF and the 7,8-dihydrodiol share the 7,8-oxide of BNF (Fig. 2) as a common precursor. The position of the phenolic hydroxyl group is based on its NMR spectrum (Table I). A typical 3- spin system for Hs, He, and H7 is present with normal values for the ortho- and meta-coupling constants. Although the same spin system would be observed for 5-hydroxy-BNF, this structure can be excluded since the proton assigned as Hs (9.55 6) is still shifted downfield due to its proximity to the carbonyl group. The signal for HS is not as far downfield as in BNF since it is para to the hydroxyl group (22). Similarly H9 is shifted downfield -0.5 ppm due to the peri hydroxyl group at position 8. The 8-hydroxy-BNF used in these studies was obtained from BNF incubated with liver microsomes from phenobarbital-treated rats.

Assignment of 5-hydroxy-BNF as a phenol is based on its mass spectrum (CI, methane gas) which showed a molecular

ion at m/e 289 (M' + 1). Although its ultraviolet spectrum (Fig. 3C) failed to show a red shift in alkaline medium, it does share BNF 5,6-oxide (Fig. 2) as a common precursor with the 5,6-dihydrodiol and can be obtained from the 5,6-dihydrodiol on acid-catalyzed dehydration. Although an adequate quan- tity of the phenol could not be obtained for an NMR spectrum, the 5-position for the hydroxyl group rather than the 6-posi- tion is tentatively favored for electronic reasons based on dehydration of the dihydrodiol or isomerization of the arene oxide.

An additional phenol of unknown structure (Table 11, phenol A) was characterized spectrally. Its ultraviolet spec- trum (Fig. 30) showed a reversible red shift in alkali and its mass spectrum (CI, isobutane gas) had a molecular ion a t m/ e 289 (M' + 1). The ultraviolet spectra of four unknown, minor metabolites are shown in Fig. 4.

Effect of Inducing Agents on the Metabolism of B N F

Fig. 5 illustrates the profiles of radioactive metabolites formed by liver microsomes from 3-methylcholanthrene- or phenobarbital-treated rats as well as a highly purified system reconstituted with cytochrome P-45Oc in the absence and presence of epoxide hydrolase. Effects of various inducers of the cytochrome P-450 system on the microsomal metabolism

by guest on February 28, 2020http://w

ww

.jbc.org/D

ownloaded from

Metabolism of a- and p-Naphthoflauones

TABLE I1 Metabolism of ['HJBNF by various rat liver microsomes and by a reconstituted monooxygenase systems

Experimental conditions were as described under "Experimental Procedures." Microsomal protein concentrations were 0.2 mg/2.0 ml and the substrate concentration was 100 nmoV2.0 ml incubation. Recovery (the percentage of the total radioactivity due to metabolism emerging from the column before the substrate in the defined metabolite fractions as compared with total radioactivity due to metabolism) was between 93-1000/0 except as indicated in note

5653

b. Individual metabolites as percentage of total metabolites

Dihydrodiols Phenols "_

Unknowns Total con- version"

5,6- 7,8- 8-OH 5-OH Phenol-A 1 2 3 4 - Microsomes

Control 2.0 23.1 55.0 4.6 9.2 1.3 0.8 1.3 2.7 4.64 (3.2)

Phenobarbital 1.5 16.1 58.6 3.9 11.8 1.2 1.0 1.2 4.7 7.10 (1.7)

3-Methylcholanthrene 11.5 63.6 6.5 6.4 1.6 6.1 1.0 1.6 1.8 20.0 (7.6)

BNF 6.7 62.0 4.9 7.4 1.5 11.0 1.5 3.0 2.8 25.9 (7.9)

Reconstituted system Cytochrome P-450~' (0.2 nmol) 4.6 17.4 11.1 0.9 9.4 12.6 Cvtochrome P-450c (0.2 nmol) 3.9 60.9 1.8 4.6 2.1 19.4 2.7 3.9 0.7 13.3

+ epoxide hydrolase (40 pg)

+ epoxide hydrolase (80 pg) Cytochrome P-45Oc (0.2 nmol) 4.4 65.4 1.3 4.1 1.6 16.8 2.5 3.1 0.6 11.3

Total metabolism denotes the percentage conversion of substrate, i.e. total radioactivity above blank which emerges from the column before the substrate. Numbers in parentheses represent rates of metabolism expressed in terms of nanomoles of substrate metabolized/nmol of cytochrome P-450/min.

* The majority of the products formed in this incubation were not detected in any of the other profiles (cf . Fig. 5).

treated rats. With these microsomes, 8-hydroxy-BNF was the major metabolite (55-59% of total metabolites), whereas the 7,8-dihydrodiol constituted only 16-23%, and the 5,6-dihydro- diol constituted about 2% of the total metabolites. On the other hand, when microsomes from 3-methylcholanthrene- or BNF-treated rats were used as a source of enzyme, the 7 8 - dihydrodiol was the major metabolite formed and constituted

w 0

Q lr 0 rn m

z >GO% of the total metabolites. The 5,6-dihydrodiol accounted

Q With the reconstituted system containing highly purified

m for 7-12% of the total metabolites, and 8-hydroxy-BNF con- stituted only 5-7% of the total metabolites.

cytochrome P-45Oc and epoxide hydrolase, the profile of me- tabolites was similar to the profiles obtained with liver micro- somes from BNF- or 3-methylcholanthrene-treated rats (Ta- ble 11). A 2-fold higher epoxide hydrolase concentration failed to block completely the formation of 5- and 8-hydroxy-BNF. When epoxide hydrolase was excluded from the incubation

, , I , , , , , mixture, very little 7&dihydrodiol was detected, 8-hydroxy- 240 260 280 300 320 340 360 380 BNF was increased by 10-fold, and 5-hydroxy-BNF was in-

WAVELENGTH (nmi creased by 2.5-fold compared to metabolism in the presence F I G . 4. Ultraviolet spectra of unknowns 1-4 of BNF. Re- of epoxide hydrolase. Also, numerous other products were

corded in the water/methanol composition at which they emerge detected which may arise from the formed arene in the from HPLC column. absence of epoxide hydrolase (cL Fig. 5). The purified system

reconstituted with cytochrome P-450b failed to metabolize of [3H]BNF are shown in Table 11. Rates of metabolism (nanomoles of BNF metabolized/nmol of cytochrome P-450/ min) were found to be linear with protein concentrations of 0.2-1.6 mg/2.0 ml incubation for microsomes from control and phenobarbital-treated rats and 0.2-0.8 mg/2.0 ml incubations for microsomes from 3-methylcholanthrene- and BNF-treated rats. Both 3-methylcholanthrene- and BNF-treatment of rats stimulated the rate of metabolism of [3H]BNF by about 2.5- fold compared to control microsomes. In contrast, phenobar- bital treatment caused about a 50% decrease in the rate of metabolism of BNF. The most dramatic difference in the metabolism of BNF by the different microsomal preparations was their regioselectivity in the formation of individual me- tabolites. In general, the profiles of metabolites formed were identical for liver microsomes from control and phenobarbital-

BNF. Structures of the ANF Metabolites-Consistent with pre-

vious reports (9, lo), we have found that liver microsomal preparations metabolize ANF to a stable arene oxide, two dihydrodiols, and two phenols along with a few minor un- known metabolites. Our data are either confirmatory or pro- vided no reason to doubt the assignments of 9-hydroxy-ANF, ANF 5,6-oxide, ANF 5,6-dihydrodiol, and 6-hydroxy-ANF, although no efforts were made to distinguish this latter phenol from its presently unknown 5-hydroxy isomer. The remaining dihydrodiol, assigned by Coombs et al. (9) and Coombs (11) as the 9,10-dihydrodiol, is in fact the 7,8-dihydrodiol' as will

Dr. S. Nesnow has advised us (personal communication) that he also has concluded that the Coombs assignment of the 9,lO-dihydro- diol is in error (see footnote 3).

- ________"____

by guest on February 28, 2020http://w

ww

.jbc.org/D

ownloaded from

5654 Metabolism of a- and ,8-Naphthoflavones

4

3

2

1

-3

2 x 9 z *

7

6

5

4

3

2

1

A PHENOBARBITAL

B 3-METHYLCHOLANTHRENE I U z a

0 10 20 30 40

~ ~~~~~~

C CYTOCHROME P450C - EPOXIDE HYDROLASE I

D CYTOCHROME P450C + EPOXIDE HYDROLASE

TIME (MINI

FIG. 5. Chromatographic separation of metabolites formed from [JH]BNF. Metabolism by liver micro- somes from phenobarbital- (A) and 3-methylcholanthrene-treated ( B ) rats, and by a highly purified system reconstituted with cytochrome P-45Oc in the absence (C) and the presence (D) of epoxide hydrolase. The percentages given in C are for peaks seen only in the absence of epoxide hydrolase and are not elsewhere tabulated. Details are given under “Experimental Procedures” as well as Table 11.

5.6-DIHYDRODIOL

HO 0

6-HYDROXY

7,8-DIHYDRODIOL

0 7-HYDROXY

4

3

2

1

O

2 x 0 2

16

14

12

10

8

6

4

2

‘0

FIG. 6. Metabolites of ANF. See also Fig. 2.

by guest on February 28, 2020http://w

ww

.jbc.org/D

ownloaded from

Metabolism of a- and ,8-Naphthoflauones 5655

TABLE I11 Metabolism of r3H]ANF by various rat liver microsomes and by a reconstituted monooxygenase system

Experimental conditions were as described under “Experimental Procedures.” The substrate and microsomal protein concentrations were 100 nmol and 0.2 mg/2.0 ml incubation, respectively. Recoveries were between 92- 96%.

Individual metabolites as nercentaee of total metabolites

Microsomes Control 22.0 ND 43.1 24.9

Phenobarbital 9.0 0.6 50.4 31.9

3-Methylcholanthrene 43.2 0.5 39.0 11.9

BNF 40.6 0.6 41.7 10.2

Reconstituted system Cytochrome P-45Oc (0.2 nmol) ND ND 86.0‘ 10.0 Cytochrome P-45Oc (0.2 nmol) + 34.8 2.2 50.3 7.3

Cytochrome P-45Oc (0.2 nmol) + 35.7 3.0 50.6 5.7 epoxide hydrolase (40 pg)

epoxide hydrolase (80 pg) -

7.3 2.7 5.85 (4.1)

6.0 2.1 10..2 (2.5)

2.6 2.8 6.34 (2.4)

2.2 4.6 8.91 (2.7)

2.5 1.5 3.63 1.8 3.7 3.45

1.8 3.2 2.58

a Definition is as described in legend of Table 11. * ND, not detected. e The 5,6-oxide and 7-hydroxy-ANF are cochromatographic under the present HPLC conditions. Although this

metabolite fraction, when formed by the microsomal preparations, appears to consist entirely of 5,6-oxide based on analysis by NMR and ultraviolet SDectroscoDy. it consists of -40% 7-hydroxy-ANF when formed by cytochrome P-

-

450~-in the absence of epoxide hy&olase.

be unequivocally shown below. The metabolism of ANF is illustrated in Fig. 6.

7,8-Dihydrodiol-Metabolite 1 and phenol 4, described by Coombs et al. (9) as the 9,lO-dihydrodiol and 10-hydroxy- ANF, are actually the 7,8-dihydrodiol and 7-hydroxy-ANF, respectively. In all probability, the large metabolite peak (Chart 3B, elution volume -7 m l ) that Nesnow and Bergman (10) assigned as the 5,6-dihydrodiol obtained with liver micro- somes from BNF-treated rats, is also the 7,8-dihydrodiol. We have isolated the dihydrodiol described by Coombs et al. (9) using liver microsomes from 3-methylcholanthrene-treated rats and have obtained identical ultraviolet and mass spectral data. Its NMR spectrum (360 MHz, acetone) was nearly identical with that reported by Coombs et al. (9) (250 MHz, dimethyl sulfoxide) on the critical benzo-ring. Chemical shifts differed by only 0.03-0.17 ppm and coupling constants were the same. Particular attention should be paid to the signal at 7.15 6 (7.10 S in our spectrum) for the benzylic vinyl hydrogen of the dihydrodiol. By comparison to the 5,6-dihydrodiol of BNF (this study) and numerous dihydrodiols of related poly- cyclic aromatic hydrocarbons (23, 24), this signal is about 0.5 ppm further downfield than would be expected in the absence of unusual deshielding factors. As pointed out by Coombs et al. (9) and ourselves (Fig. l), the signal for HIo in ANF appears at unusually low field. Therefore the benzylic vinyl hydrogen at 7.10-7.15 6 in the dihydrodiol is best assigned at position 10 consistent with a 7,8-dihydrodiol structure. As expected for this structure, a small value of 5J6,10 (-) 1 Hz is observed. Both methine hydrogens (H, and H8) are at the usual posi- tions, and the value of J7,8 = 10 Hz requires a trans-pseudo- diequatorial conformation.

Dehydration of the 7,8-dihydrodiol with acid provided a single major product which was identical with synthetic 7- hydroxy-ANF based on its ultraviolet spectrum (identical with the spectrum reported by Coombs et al. (9) for the dehydra- tion product assigned as 10-hydroxy-ANF), retention time on HPLC (identical with ANF 5,6-oxide, cf. Table I11 and Fig. 7), and NMR spectrum (500 MHz, acetone). This phenol is also formed at the expense of the 7,8-dihydrodiol by the reconsti-

2.0

1.5

1 .o

0.5

e 0

X - 6 2.0

1.5

1 .o

0.5

B PHENOBARBITAL I

0 10 20 30 40 TIME (MINI

I

ned FIG. 7. ChromatoeraDhic seDarations of metabolites fc ” Dm from [’HIANF. Liver microsomes from (A) BNF-treated rats and ( B ) phenobarbital-treated rats.

tuted system in the absence of epoxide hydrolase (see later). Comparison of our NMR spectrum (360 MHz, dimethyl sulf- oxide) of the diol dehydration product with the similarly derived metabolite 4 (250 MHz, dimethyl sulfoxide) described by Coombs et al. (9) as 10-hydroxy-ANF indicates that chem-

by guest on February 28, 2020http://w

ww

.jbc.org/D

ownloaded from

5656 Metabolism of a- and fi-Naphthoflauones

ical shifts differ by less than 0.05 ppm on the 7,8,9,10-benzo- ring and coupling constants are the same. In the Coomb’s assignment, H7 fails to show the expected upfield shift for a hydrogen para to a phenolic hydroxyl group when compared to the spectrum of ANF in dimethyl sulfoxide. In our assign- ment3 of the dehydration product as 7-hydroxy-ANF, both HR and HIO show the expected (22) upfield shift (>0.4 ppm) for hydrogens ortho and para to a phenolic hydroxyl group.

5,6-0xide Hydr~tion-[~H]ANF 5,6-oxide was isolated from large scale incubations with liver microsomes from 3-methyl- cholanthrene-treated rats as described under “Experimental Procedures.” Incubations (15 min, 37 “C) with homogeneous epoxide hydrolase contained up to 0.6 mg of protein, 300 pmol of Tris buffer (pH 8.9 at 37 “C), and 100 nmol of 5,6-oxide (in 0.05 ml of acetonitrile) to reach a final volume of 1.0 ml. Unreacted 5,6-oxide and product dihydrodiol were extracted into acetone-ethyl acetate as described for other metabolites. The amount of dihydrodiol formed was determined by liquid scintillation spectroscopy after separation from the 5,6-oxide by HPLC under conditions used for analysis of the metabolism of ANF. Metabolism was linear with protein up to 0.4 mg of protein/ml, and the rate of metabolism was 1.0 nmol of 5,6- dihydrodiol formed/mg of protein/min. An incubation minus enzyme served as control.

Effects of Inducing Agents on the Metabolism of ANF Comparative metabolism of [3H]ANF by liver microsomes

from control and treated rats is shown in Table 111, and two representative metabolite profiles are shown in Fig. 7. In contrast to the metabolism of BNF, all three inducers of the cytochrome P-450 system caused a 34-41% decrease in the rate of metabolism of ANF relative to microsomes from con- trol rats when the data were expressed per nmol of cytochrome P-450. Compared to the metabolism of BNF, control micro- somes metabolized ANF at a slightly higher rate. The three major metabolites of ANF were the 7&dihydrodiol, the 5,6- oxide, and 6-hydroxy ANF. Their relative amounts were de- pendent on the source of microsomes used. The sum of the 5,6-oxide and the 6-phenol represented a major pathway of metabolism (5142% of total metabolites), while the 5,6-di- hydrodiol was the least significant metabolite (<1%) formed by each of the four microsomal preparations. No particular significance can be attached to the ratio of phenol to arene oxide since minor variations in workup could result in different extents of isomerization of the arene oxide to the phenol (25). With microsomes from 3-methylcholanthrene- and BNF- treated rats, the 7,8-dihydrodiol was also a major product, representing 41-43% of the total metabolites. In contrast, with microsomes from control and phenobarbital-treated rats, this metabolite accounted for only 22 and 9% of the total metab- olites. Several minor metabolites were also formed, including

The purified system reconstituted with cytochrome P-45Oc and epoxide hydrolase gave results nearly identical with those obtained with liver microsomes from 3-methylcholanthrene- or BNF-treated rats. Doubling the concentration of epoxide hydrolase had little effect, whereas the 5,6- and 7,8-dihydro- diols were not formed when epoxide hydrolase was omitted from the incubation. In the absence of epoxide hydrolase, the 5,g-oxide fraction increased proportionally to the amount of dihydrodiols lost, a consequence mainly of the fact the 7- hydroxy ANF cochromatographs with the 5,6-oxide. The pur-

9-hydroxy ANF (2-7%).

Dr. R. Roth has also concluded (personal communication) that the dehydration product is 7-hydroxy ANF based on comparison of the NMR spectrum of his synthetic product with the reported spec- trum (9).

S e d system reconstituted with cytochrome P-450b gave no detectable conversion of substrate.

Effects of Antibodies against Cytochrome P-450 Isozymes on the Liver Microsomal Metabolism of ANF and BNF Antibodies directed against cytochromes P-450a, P-450b, P-

450c, and P-450d were used to determine the contribution of these isozymes in the overall metabolism of ANF and BNF by liver microsomes. Cytochromes P-450b and P-45Oc are the major rat liver isozymes induced by phenobarbital and 3- methylcholanthrene, respectively (18, 19). Cytochrome P- 450d, the major isosafrole-induced isozyme, is also induced by 3-methylcholanthrene but not by phenobarbital treatment of rats (17, 26). Cytochrome P-450a is an isozyme modestly inducible by phenobarbital and 3-methylcholanthrene (18, 19). Total metabolism of ANF by microsomes from pheno- barbital-treated rats was not inhibited by antibodies directed against cytochromes P-450a, P-450b, or P-450d (Table IV). Since cytochrome P-450b is immunochemically identical with cytochrome P-450e (15), a minor isozyme inducible by pheno- barbital, neither of these hemoproteins play a significant role in the metabolism of ANF by microsomes from phenobarbital- treated rats. The lack of inhibition of the metabolism of ANF by anti-P-450b is consistent with the observation that purified cytochrome P-450b does not metabolize this substrate to any significant extent. Furthermore, neither cytochrome P-450d nor P-45Oc participate in the metabolism of ANF catalyzed by microsomes from phenobarbital-treated rats because anti- P-450d, which inherently cross-reacts and inhibits the cata- lytic activity of cytochrome P-45Oc (20), failed to inhibit metabolism of ANF.

Metabolism of ANF catalyzed by liver microsomes from 3- methylcholanthrene-treated rats was not inhibited by anti-P-

TABLE IV Immune complex inhibition of the metabolism of [’HIANF and

[3H]BNF catalyzed by rat liver microsomes Liver microsomes (0.3 nmol of cytochrome P-450) were preincu-

bated with the appropriate IgG fraction at 22 “C for 10 min in phosphate-buffered saline (pH 7.4). All assay tubes were brought to the same final concentration of IgG with control IgG. After this preincubation, the samples were chilled in ice, cofactors and buffer were added, and incubations were performed as described under “Experimental Procedures.”

Microsomes Antibody

Metabolism of ANF

Phenobarbi- tal

3-Methyl- cholan- threne

Metabolism of BNF

Phenobarbi- tal

3-Methyl- cholan- threne

Control Anti-P-450a Anti-P-450b Anti-P-450b Anti-P-450d Control Anti-P-450a Anti-P-450c(-d) Anti-P-450c(-d) Anti-P-450d

Control Anti-P-450a Anti-P-450b Control Anti-P-450a Anti-P-450c(-d) Anti-P-450c(-d) Anti-P-450d

::%:! Metabo- Control 450 lism activity

nmol/min/ R nmol

12 3.7 100 12 3.8 103 4 4.2 114

12 3.9 105 12 3.5 95 12 3.2 100 12 3.0 94 4 2.8 88

12 2.7 84 12 2.3 72

12 2.2 100 12 1.9 86 4 1.9 86

12 8.8 100 12 7.1 81 4 2.1 24

12 1.6 18 12 1.4 16

by guest on February 28, 2020http://w

ww

.jbc.org/D

ownloaded from

Metabolism of a- and P-Naphthoflavones 5657

450a and only modestly inhibited (16-28%) by anti-P-450c(-d) or anti-P-450d (Table IV), despite the fact that these three isozymes collectively constitute greater than 90% of total cytochrome P-450 in these microsomes (17, 26). While a reconstituted system fortified with cytochrome P-45Oc does metabolize ANF (Table 111)) it is clear from the antibody results that other uncharacterized cytochrome P-450 isozymes in liver microsomes from 3-methylcholanthrene-treated rats are more important than is cytochrome P-45Oc in the total metabolism of ANF. However, the change in the profile of metabolites from ANF following 3-methylcholanthrene-treat- ment of rats (cf Table 111) is due primarily to the marked induction of cytochrome P-45Oc (Fig. 8A). When the propor- tion of individual metabolites of ANF formed by these micro- somes is analyzed in the presence of anti-P-450c(-d) or anti-P- 450d (which recognizes and inhibits cytochrome P-450c), the profiie of metabolites mimics that observed with microsomes from control rats. In other words, the uninhibited metabolism of ANF by microsomes from 3-methylcholanthene-treated rats (72-84% of total metabolism) reflects the metabolite pattern obtained with microsomes from control rats.

Metabolism of BNF by microsomes from phenobarbital- treated rats was not significantly inhibited by anti-P-450a or anti-P-450b) as was observed when ANF was used as substrate (Table IV). Purified cytochrome P-450b also failed to metab- olize BNF in a reconstituted system. In marked contrast to the lack of dramatic inhibition of the metabolism of ANF (which is not induced by 3-methylcholanthrene-treatment of rats), metabolism of BNF by these microsomes (induced 2.5- fold) was markedly inhibited (82%) by anti-P-450c(-d). Anti- P-450d, which also recognizes cytochrome P-450c, caused no

75 A METABOLITES OF ANF

I 0 CONTROL MICROSOMES I5METHYLCHOLANTHRENE-TREATED CZI TREATED + anti P450c(-dl

25

z t

s 7.8.OIHYORO. S.OOIHYDR0- DIOL DIOL

n l M k m L

OHYOROXY OHYDUOXY UNKNOWNS

% ; 77- E METABOLITES OF BNF

FIG. 8. The effect of anti-P-46Oc(-d) on the per cent distri- bution of individual metabolites formed from (A) [*WANF and (I?) [‘HIBNF. Details are given under “Experimental Procedures” and in Table IV.

additional inhibition of metabolism than was observed with anti-P-450c(-d), indicating that cytochrome P-450d does not contribute to the metabolism of BNF by microsomes from 3- methylcholanthrene-treated rats. The marked inhibition of the metabolism of BNF by anti-P-450c(-d) also resulted in a change in the metabolite profiie (Fig. 8B). The residual cata- lytic activity of the microsomes from 3-methylcholanthrene- treated rats in the presence of anti-P-450c(-d) resulted in a metabolite pattern which was virtually identical with that seen with liver microsomes from untreated rats. This result, coupled with the observation that a reconstituted system fortified with purified cytochrome P-45Oc and epoxide hydro- lase resulted in a metabolite profile which was virtually iden- tical with that obtained with liver microsomes from 3-meth- ylcholanthrene-treated rats (cf Table 11), indicates that cy- tochrome P-45Oc is the predominant isozyme catalyzing the metabolism of BNF by liver microsomes from 3-methylchol- anthrene-treated rats. Notably, treatment of these rats with either BNF or 3-methylcholanthrene results in liver micro- somal preparations which have similar compositions of the various cytochromes P-450 (26). In both cases the major component (>70%) is cytochrome P-45Oc (26). Whether pur- ified from livers of BNF- or 3-methylcholanthrene-treated rats, the hemeprotein has been shown to be virtually identical by a number of criteria (27).

DISCUSSION

Quantitative evaluation of the effect of phenobarbital treat- ment of rats on the metabolism of ANF and BNF by liver microsomes has revealed a 40-50% decrease compared to rates of metabolism (nanomoles of metabolites/nmol of cytochrome P-450) by microsomes from control rats. The depressed rate of metabolism of either substrate after treatment of rats with phenobarbital is in concurrence with the fact that cytochrome P-450b increases from <5 to >50% of total microsomal cyto- chrome P-450 on phenobarbital treatment (18, 19) and that the highly purified system reconstituted with cytochrome P- 450b failed to metabolize either substrate. The lack of inhibi- tion of the metabolism of ANF and BNF by anti-P-450b in microsomes from phenobarbital-treated rats provided addi- tional proof that cytochrome P-450b (and cytochrome P-450e) does not metabolize either substrate to any significant extent. Antibodies directed against three other cytochrome P-450 isozymes (P-450a, P-450c, and P-450d) failed to inhibit metab- olism in these microsomes. These results indicate that un- characterized isozymes of cytochrome P-450 are highly effec- tive in the metabolism of ANF and BNF by microsomes from phenobarbital-treated rats.

Prior treatment of rats with either 3-methylcholanthrene or BNF results in a -140% increase in the rate of metabolism (nanomoles of product/nmol of cytochrome P-450) of BNF and in a 41% decrease in the rate of metabolism of ANF compared to liver microsomes from control rats. In contrast to the observation that phenobarbital-treatment of rats did not cause any changes in the relative proportion of individual ANF or BNF metabolites compared to microsomes from control rats, 3-methylcholanthrene or BNF-treatment of rats caused a substantial change in metabolite distribution. For each substrate very similar profiles of products are obtained with microsomes from BNF- or 3-methylcholanthrene-treated rats and with a purified system reconstituted with cytochrome P-45Oc and epoxide hydrolase. Evidence supporting the con- cept that cytochrome P-45Oc is primarily responsible for the change in metabolite pattern observed with these microsomes was obtained from antibody studies with anti-P-450e(-d). The distribution of ANF and BNF metabolites formed by micro- somes from 3-methylcholanthrene-treated rats in the presence

by guest on February 28, 2020http://w

ww

.jbc.org/D

ownloaded from

5658 Metabolism of a- and /3-Naphthoflauones

of anti-P-450c(-d) was virtually identical with that obtained with microsomes from control rats. Since overall metabolism of ANF and BNF by microsomes from 3-methylcholanthrene- treated rats was inhibited to markedly different extents (14- 28% and 82%, respectively), cytochrome P-450 isozymes other than cytochrome P-45Oc obviously play a major role in the metabolism of ANF but not BNF by these microsomes. The use of specific antibodies (18-20, 26) has provided a particu- larly powerful tool for the evaluation of the contribution of individual cytochrome P-450 isozymes to the overall liver microsomal metabolism of ANF and BNF.

All the major metabolites of both flavonoids were formed on the naphthalene ring, and as yet metabolites have not been detected on their phenyl substituents (Figs. 2 and 6). Most of the metabolism of BNF (8048%) proceeds via its 5,6- and 7,8- oxides, mainly the latter, and the corresponding phenols formed by isomerization (25) and dihydrodiols formed by the action of epoxide hydrolase were isolated. The ratio of 8- hydroxy-BNF to 7,8-dihydrodiol formed proved quite inter- esting. With microsomes from control- or phenobarbital- treated rats, 70-78% of the arene oxide isomerizes to the phenol and only 22-30% is trapped as the dihydrodiol by epoxide hydrolase. In contrast, with microsomes from 3-meth- ylcholanthrene- or BNF-treated rats, >90% of the 7,8-oxide is trapped as the 7,8-dihydrodiol by epoxide hydrolase and 4 0 % isomerizes to the phenol. It is very tempting to speculate that predominantly a single enantiomer of the 7,8-oxide is formed by the cytochrome P-45Oc isozyme in liver microsomes from 3-methylcholanthrene- and BNF-treated rats, and that this enantiomer is an excellent substrate for epoxide hydiolase. Consistent with this proposal is the fact that the presence of epoxide hydrolase decreases &hydroxy BNF to a trace metab- olite in the reconsituted system containing cytochrome P-45Oc (Table 11). Isozymes other than cytochrome P-45Oc in liver microsomes from control and phenobarbital-treated rats pre- sumably form a substantial amount of the opposite enantio- mer which is proposed to be a poor substrate for epoxide hydrolase. Several examples of high enantioselectivity by epoxide hydrolase have now been documented for arene oxide and epoxide substrates (28, 29).

The 5,6-dihydrodiol of BNF proved to be quite unusual in that it adopts a rather novel pseudo-diaxial conformation for its hydroxyl groups (Fig. 9). Presumably adverse steric inter- actions as well as possible dipole-dipole interactions between the 5-hydroxyl group and the carbonyl oxygen force the 5- hydroxyl group out of the plane of the molecule. Previously a pen methyl (34-36) or fluorine substituent (37, 38) and the proximity to a bay region (24) in a polycyclic aromatic hydro- carbon had been noted to cause such conformational changes. Factors which cause conformational changes of dihydrodiols are critically important in chemical carcinogenesis by the hydrocarbons (37) as they affect the extent to which bay- region diol epoxides, the ultimate carcinogens (39), are formed.

Previous studies of the metabolism of ANF have proved quite interesting in that a stable 5,g-arene oxide had been identified as a metabolite (9-11). Part of this high stability must stem from the carbonyl group. Examples of such stabi- lization are known from the chemistry of substituted benzene oxides (40). The second reason that ANF 5,6-oxide survives the incubation conditions is that it is a remarkably poor substrate for epoxide hydrolase. (1 nmol of dihydrodiol

H -0‘ FIG. 9. Molecular model of the preferred pseudo-&axial con-

formation of BNF 5,6-&hydro&ol generated by the program XRAY (30). The structure shown is based on crystallographic data for 3’,5,5’,6-tetramethoxyflavone (31) and a dihydrodiol of apolygclic hydrocarbon (32). The distance between the carbonyl oxygen and hydroxyl oxygen is 2.8 A. In the pseudo-diequatorial conformation (not shown), chis distance is reduced to only 1.9 8, which is much less than the sum of the van der Waals radii for oxygen (33) . Hence, steric hindrance between these oxygen atoms would be very severe in the pseudo-diequatorial conformation.‘

FIG. 10. Application of a steric model for the catalytic binding site of cytochrome P-45Oc to the metab- olism of ANF. The basic minimal boundary (A) of the binding site and the position of oxygenation has been de- duced through studies of the metabolism of benzo[a]pyrene (43). Attempts to form ANF 9,lO-oxide result in the phenyl sub- stituent having to reside in a restricted area ( B ) . A single enantiomer of the 5,6- oxide (C) is predicted to be formed by the model. Unpublished studies of the metabolism of dibenz[a,j]anthracene to its 3,4-dihydrodiol have indicated that the region occupied by the phenyl sub- stituent in ANF 7,d-oxide (D) is an al- lowed region. Thus, the minimal binding site has been enlarged (D) to accom- modate the phenyl substituent and to indicate how the ANF 7,8-oxide could be formed.

A. SITE MODEL

C. ANF 5,6-OXIDE

6. ANF 9’10-OXIDE

D. ANF 7,8-OXIDE

by guest on February 28, 2020http://w

ww

.jbc.org/D

ownloaded from

Metabolism of a- and P-Naphthoflauones 5659

formed/mg of epoxide hydrolase/min). It is, in fact, the poor- est arene oxide substrate yet examined with the purified enzyme (41). The proximate carbonyl group may be respon- sible for its poor activity (42).

The report by Coombs et al. (9) that the 9,10-dihydrodiol was the predominent metabolite of ANF with liver micro- somes from 3-methylcholanthrene-treated rats had initially perplexed us as the precursor 9,lO-arene oxide seemed an improbable metabolite based on our steric model (43-45) for the catalytic binding site of cytochrome P-45Oc. Re-evaluation of the structure of this metabolite has established that it is actually the 7,B-dihydrodiol whose formation is consistent with our view of the regiospecifkity of cytochrome P-450~ (Fig. 10). The purified monooxygenase system reconstituted with cytochrome P-45Oc and epoxide hydrolase forms this dihydrodiol to the extent of 36% of the total metabolites. Likewise, the 7,8-dihydrodiol constituted 41-43% of total me- tabolites of ANF when microsomes from 3-methylcholan- thene- and BNF-treated rats were used. On the other hand, we do not understand the mechanism by which 9-hydroxy- ANF is formed. At best it is only a trace metabolite when formed by the purified system reconstituted with cytochrome P-45Oc (Table 111). Its extent of formation is not affected by the presence of epoxide hydrolase. Perhaps it arises by some form of direct hydroxylation which does not involve an arene oxide intermediate (46).

Acknowledgments-We are deeply indebted to Drs. S. Nesnow and R. Roth for their helpful exchanges of information and for the samples of synthetic 7- and 9-hydroxy ANF. We also thank Drs. J. M. Sayer and D. Michaud at the National Institutes of Health for the computer-generated drawings.

1.

2.

3.

4.

5. 6.

7.

8.

9.

10. 11. 12.

13.

14. 15.

16.

17.

18.

REFERENCES

Wiebel, F. J. (1980) in Carcinogenesis: Modifiers of Chemical Carcinogenesis (Slaga, T. J., ed) Vol. 5, pp. 57-84, Raven Press, New York

Huang, M. T., Johnson, E. F., Muller-Eberhard, A., Koop, D. R., Coon, M. J., and Conney, A. H. (1981) J. Biol. Chem. 256,

Lasker, M. J., Huang, M. T., and Conney, A. H. (1982) Science (Wash. D. C.) 216, 1419-1421

Digiovanni, J., Slaga, T. J., Berr, D. L., and Juchau, M. K. (1980) in Carcinogenesis: Modifiers of Chemical Carcinogenesis (Slaga, T. J., ed) Vol. 5, pp. 145-168, Raven Press, New York

Wattenberg, L. W. (1978) Adu. Cancer Res. 26, 197-226 Stegeman, J . J., and Woodin, B. K. (1980) Biochem. Biophys.

Res. Commun. 95, 328-333 Thakker, D. R., Levin, W., Buening, M., Yagi, H., Lehr, R. E.,

Wood, A. W., Conney, A. H., and Jerina, D. M. (1981) Cancer Res. 41, 1389-1396

Nesnow, S., Bergman, H., and Sovocool, W. (1980) Proc. Am. Assoc. Cancer Res. 21,63

Coombs, M. M., Bhatt, T. S., and Vose, C. W. (1981) Carcinogen- esis (Lond.) 2, 135-140

Nesnow, S., and Bergman, H. (1981) Cancer Res. 41,2621-2626 Coombs, M. M. (1982) Carcinogenesis (Lond.) 3,229-230 Lu, A. Y. H., and Levin, W. (1972) Biochem. Biophys. Res.

Lowry, 0. H., Rosebrough, N. J., Farr, A. L., and Randall, R. J.

Omura, T., and Sato, R. (1964) J. Biol. Chem. 239, 2379-2385 Ryan, D. E., Thomas, P. E., and Levin, W. (1982) Arch. Biochem.

Yasukochi, Y., and Masters, B. S. S. (1976) J. Biol. Chem. 251,

Ryan, D. E., Thomas, P. E., and Levin, W. (1980) J. Biol. Chem.

Thomas, P. E., Korzeniowski, D., Ryan, D. E., and Levin, W.

10897-10901

Commun. 46, 1334-1339

(1951) J. Biol. Chem. 193,265-275

Biophys. 216,272-288

5337-5344

255, 7941-7955

(1979) Arch. Biochem. Biophys. 192,524-532

19. Thomas, P. E., Reik, L. M., Ryan, D. E., and Levin, W. (1981) J. Biol. Chem. 256,1044-1052

20. Reik, L. M., Levin, W., Ryan, D. E., and Thomas, P. E. (1982) J . Biol. Chem. 257,3950-3957

21. Hanzlik, R. P., Wiley, R. A,, and Gillesse, T. J. (1979) J. Labelled Compd. Radiopharm. 16, 523-529

22. Becker, E. D. (1969) High Resolution NMR, pp. 73-76, Academic Press, New York

23. Jerina, D. M., Daly, J. W., Witkop, B., Zaltzman-Nirenberg, P., and Udenfriend, S. (1970) Biochemistry 9, 147-156

24. Jerina, D. M., Selander, H., Yagi, H., Wells, M. C., Davey, J. F., Mahadevan, V., and Gibson, D. T. (1976) J . Am. Chem. SOC.

25. Jerina, D. M., Yagi, H., and Daly, J . W. (1973) Heterocycles 1,

26. Thomas, P. E., Reik, L. M., Ryan, D. E., and Levin, W. (1982), in

27. Guengerich, F. P., and Martin, M. V. (1980) Arch. Biochem. press

Biophys. 205,365-379 28. Levin, W., Buening, M. K., Wood, A. W., Chang, R. L., Kedzierski,

B., Thakker, D. R., Boyd, D. R., Gadaginamath, G. S., Arm- strong, R. N., Yagi, H., Karle, J. M., Slaga, T. J., Jerina, D. M., and Conney, A. H. (1980) J. Biol. Chem. 255,9067-9074

29. Watabe, T., Ozawa, N., and Yoshikawa, K. (1981) Biochem. Pharmacol. 30,1695-1698

30. Feldmann, R. J., Bacon, C. R. T., and Cohen, J. S. (1973) Nature (Lond.) 244, 113-115

31. Ting, H.-Y., Watson, W. H., and Dominguez, X. A. (1972) Acta Crystallogr. Sect. E Struct. Crystallogr. Cryst. Chem. 28,1046- 1051

32. Zacharias, D. E., Glusker, J. P., Fu, P. P., and Harvey, R. G. (1979) J. Am. Chem. SOC. 101,4043-4051

33. Pauling, L. (1960) The Nature of the Chemical Bond, pp. 449- 504, Cornell University Press, Ithaca, New York

34. Jerina, D. M., Sayer, J. M., Thakker, D. R., Yagi, H., Levin, W., Wood, A. W., and Conney, A. H. (1980) in Carcinogenesis: Fundamental Mechanisms and Environmental Effects (Pull- man, B., Ts’O, P. 0. P., and Gelboin, H., eds) pp. 1-12, D. Reidel Publishing Co., Dordrecht, Holland

35. Yang, S. K., Chou, M. W., and Fu, P. P. (1980) in Carcinogenesis: Fundamental Mechanisms and Environmental Effects (Pull- man, B., Ts’O, P. 0. P., and Gelboin, H. eds) pp. 143-156, D. Reidel Publishing Co., Dordrecht, Holland

36. Sims, P. (1980) in Carcinogenesis: Fundamental Mechanisms and Environmental Effects (Pullman, B., Ts’O, P. 0. P., and Gelboin, H., eds) pp. 33-42, D. Reidel Publishing Co., Dor- drecht, Holland

37. Buhler, D. R., Unlu, F., Slaga, T. J., Newman, M. S., Levin, W.,

4783 Conney, A. H., and Jerina, D. M. (1982) Cancer Res. 42,4779-

38. Chiu, P. L., Fu, P. P., and Yang, S. K. (1982) Biochem. Biophys. Res. Commun. 106. 1405-1411

39. Nordqvist, M., Thakker, D. R., Yagi, H., Lehr, H. E., Wood, A. W., Levin, W., Conney, A. H., and Jerina, D. M. (1980) in Molecular Basis of Environmental Toxicity (Bhatnagar, R. S., ed) pp. 329-357, Ann Arbor Science Publishers, Ann Arbor, Michigan

40. Chiasson, B. A., and Berchtold, G. A. (1977) J. Org. Chem. 42, 2008-2009

41. Lu, A. Y. H., Jerina, D. M., and Levin, W. (1977) J . Biol. Chem.

42. Oesch, F., Kaubisch, N., Jerina, D. M., and Daly, J. W. (1971) Biochemistry 10,4858-4866

43. Jerina, D. M., Michaud, D. P., Feldmann, R. S., Armstrong, R. N., Vyas, K. P., Thakker, D. R., Yagi, H., Ryan, D. E., Thomas, P. E., and Levin, W. (1982) in Microsomes, Drug Oxidations and Drug Toxicity (Sato, R., and Kato, R., eds) pp. 195-201, Japan Scientific Societies Press, Tokyo

44. van Bladeren, P. J., Armstrong, R. N., Cobb, D., Thakker, D. H., Ryan, D. E., Thomas, P. E., Sharma, N. D., Boyd, D. R., Levin, W., and Jerina, D. M. (1982) Biochem. Biophys. Res. Commun.

45. Yagi, H., and Jerina, D. M. (1982) J. Am. Chem. SOC. 104,4026-

46. Tomaszewski, J. E., Jerina, D. M., and Daly, J. W. (1975) Bio-

98,5988-5996

267-326

252, 3715-3723

106,602-609

4027

chemistry 14, 2024-2031

by guest on February 28, 2020http://w

ww

.jbc.org/D

ownloaded from

JerinaK P Vyas, T Shibata, R J Highet, H J Yeh, P E Thomas, D E Ryan, W Levin and D M

microsomes and highly purified reconstituted cytochrome P-450 systems.Metabolism of alpha-naphthoflavone and beta-naphthoflavone by rat liver

1983, 258:5649-5659.J. Biol. Chem. 

  http://www.jbc.org/content/258/9/5649.citation

Access the most updated version of this article at

 Alerts:

  When a correction for this article is posted• 

When this article is cited• 

to choose from all of JBC's e-mail alertsClick here

  http://www.jbc.org/content/258/9/5649.citation.full.html#ref-list-1

This article cites 0 references, 0 of which can be accessed free at

by guest on February 28, 2020http://w

ww

.jbc.org/D

ownloaded from