The metabolism of benzo[a]pyrene phenols by rat liver microsomal fractions

ARCHIVES OF BIOCHEMISTRY Vol. 212, No. 1, November,

AND BIOPHYSICS pp. 136-146, 1981

The

RUSSELL Lkpartment

Metabolism of Benzo[a]pyrene Phenols by Rat Liver Microsomal Fractions

A. PROUGH,’ of Biochemistry, 5323 Harry Received

May

YUKI

SAEKI,

The University Hines Boukvard,

AND

of Terns Lkzllas,

11, 1981, and in revised

JORGE

Health science Texas 75.235

form

July

CAPDEVILA Center

at LkxUas,

‘7, 1981

The oxidative metabolism of benztialpryrene (BEalP) phenols catalyzed by liver microsomes in vitro leads to multiple products. High-pressure liquid chromatography analysis of the organic-soluble products formed indicates that regardless of the animal pretreatment regime, 3-hydroxy-NolP is metabolized to the 3,6-quinone and to a hydroxylated derivative tentatively identified as 3,9-dihyroxy-qa]P. However, the distribution of products obtained with I)-hydroxy-Na]P varied with animal pretreatment. A maximum of three distinct metabolites was obtained when the g-phenol was metabolized in vitro with microsomes from phenobarbital-pretreated rats and the tentative 3,9-dihydroxy derivative was a common metabolite for all pretreatment regimes. Physical characterization, including mass spectrometry, indicates that all three products have an extra oxygen atom incorporated into their molecular structure from molecular oxygen. Studies utilizing specific inhibitors of the cytochrome P-450-dependent monooxygenase clearly suggest that the formation of dihydroxy or phenol-oxide derivatives is catalyzed by the hemoprotein, cytochrome P-450. These metabolites of the benzdalpyrene phenols are most likely related to the putative phenol-oxides of benzo[alpyrene which have been demonstrated to alkylate DNA and protein. Repetitive scan difference spectrophotometric analysis of incubation mixtures containing rat liver microsomes, 3- or 9-hydroxy-B[a]P, NADPH, and oxygen shows the conversion of the phenols into products which absorb in the region from 400 to 500 nm. During and after the steady state of the reaction, it can be seen that certain of the hydroxy compounds produced are in equilibrium with their respective quinone form and may be involved in an oxygen-coupled redox cycle.

The realization that the formation of an ultimate carcinogenic form of benzo[alpyrene (B[a]P),2 the truns-7,&dihydro7,8-diol-9,10-epoxy-B[a]P, requires several metabolic steps (1) has increased interest in the further metabolism of the various dihydrodiol, quinone, and phenol products which are derived from this model polycyclic aromatic hydrocarbon (2). Subsequent studies have provided molecular detail regarding the chemical and enzymatic nature of the reactions to form diol-ep-

oxides of B[a]P (3, 4). In addition, several reports have indicated that the 3- and 9phenols of B[a]P are metabolized to yield products capable of binding to cellular nucleophiles such as DNA or protein and of inducing mutation in bacteria (5-7). To date, little information is available relative to the chemical or enzymic nature of the metabolism of these B[u]P phenols. This report will describe the complexity of the chemical and enzymatic properties of the NADPHand oxygen-dependent phenol monooxygenase activity of rat liver microsomal fractions.

’ To whom correspondence should be addressed. ’ Abbreviations used: Ha]P. benzaalpyrene; BF. benzoflavone; PB, phenobarbital; TCPO, trichloropropene-2,3-oxide; HPLC, high-pressure liquid chromatography. 0003-9861/81/130136-11$02.00/O Copyright All righta

Q 1981 by Academic Press. Inc. of reproduction in any form reserved.

MATERIALS Liver Dawley 136

AND

METHODS

microsomal protein from male Spraguerats were prepared as described by Prough

BENZqajPYRENE

PHENOL

et al. (8). Phenobarbital in saline (80 mg/kg body wt), 5,6-benzoflavone in corn oil (60 mg/kg body wt), or 3-methylcholanthrene in corn oil (20 mg/kg body wt) were administered daily for 4 days and the animals were starved for 18 h prior to sacrifice. Authentic B[u]P metabolites were obtained from the National Cancer Institute Carcinogenesis Research Program, Bethesda, Maryland. [7,10-‘4C~enzo[olpyrene was purchased from Amersham/Searle. Arlington Heights, Illinois. The radiolabeled 3- and g-phenols were prepared by incubating B[ulp with 1 mg/ml liver microsomes from 5.6~benzoflavone-pretreated rats, NADPH, and oxygen. The metabolites were extracted with ethyl acetate and purified using a pBondapak Cl8 HPLC column (Waters Associates, Milford, Mass.) with a 30-min methanol:water gradient (6089%) (8). The fraction which eluted at the same position as 3-hydroxybenzo[ulpyrene was rechromatographed as described above; 99% of the radioactivity chromatographed with the peak with a retention time identical to the 3-phenol. The [i4C]-9,10-dihydrodiol of Qalp was converted to 9-hydroxy-[‘*mujP by the method of Sims (9). The resultant radiolabeled O-phenol was further purified as described above; it had an HPLC retention time idential to authentic 9hydroxy-@a]P (>98% purity). All other reagents were of the highest purity commercially available. The standard reaction mixture utilized in the study contained 50 mM Tris buffer, pH 7.5, 150 rnM KCI, 5 mM MgClz, 2 mM sodium isocitrate, 1 II-l/ml of isocitrate dehydrogenase, and liver microsomes (0.2-0.5 mg/ml) at 3’7°C. Repetitive scan difference spectrophotometry was performed as described previously (8, 10). The concentration of 3- and 9-hydroxy-B[o]P used was 50 pM except for experiments evaluating the concentration dependence of the reaction. The metabolism of the 3-phenol was measured spectrophotometrically by monitoring the increase in absorbance at 448 nm. This wavelength corresponds to the absorbance maximum of the 3,6-dihydroxy (hydroquinone) derivative of B[a]P, an extinction coefficient of 21,000 Me' cm-’ was assumed. The metabolism of the g-phenol was monitored spectrophotometrically by measuring the increase in absorbance at 436 nm. This wavelength corresponds to the absorbance maximum of metabolite A; an extinction coefficient of 26,000 M-' cm-’ was used. This value was approximated using the absorbance of the isolated metabolite and the specific radioactivity of the starting 9phenol substrate. The metabolites of the 3- and g-phenol were isolated by HPLC. The 14C-labeled phenols (approx 3.2 mCi/mmol) were added to the standard reaction mixture at 37°C and aliquots taken at various time points between 0 and 10 min. The reaction mixtures were extracted with chilled ethyl acetate, the ethyl acetate phase was filtered, and the organic phase was evaporated to dryness under a nitrogen stream. After

METABOLISM

137

HPLC isolation (as described above), the various metabolites were quantitated by liquid scintillation techniques. The specific enzyme activity was obtained by using the linear portion of several time points between 0 and 10 min. The metabolites of the 3- and g-phenols were chromatographed twice and the mass spectra were obtained using a Finnegan Model 4021 gas chromatograph-mass spectrograph using direct probe analysis with programmed temperature accessory. The absorbance spectra of the metabolites were recorded on a Cary 14 dual beam spectrophotometer and uncorrected fluorescence spectra were recorded using an Aminco Bowman Spectrophotofluorometer. The experiments measuring oxygen-18 incorporation from ‘*Oz were performed in a sealed flask containing the standard reaction mixture with 1 mg/ml liver microsomal protein from phenobarbital-pretreated rata. The system was purged of 160z by repetitive evacuation of the closed reaction flask and readdition of anaerobic nitrogen through a rubber septum. Finally, 20% of the Nz atmosphere was removed using a gas-tight syringe and an equal volume of ‘*Oz gas added to the flask. After initiation of the reaction by addition of an anaerobic solution of NADPH, an aliquot of the reaction mixture was taken at 6 min using a gas-tight syringe and was mixed rapidly with ethyl acetate. The B[u]P metabolites were isolated by HPLC as described previously (8) and rechromatographed on a PBondapak NH2 column using an isocratic separation with hexane: methylene chloride:acetonitrile (60:38.5:1.5). After evaporation, the samples were analyzed on a Finnegan Model 4021 gas chromatograph-mass spectrograph as described above. The percentage enrichment was determined by comparing M and M + 2 for the metabolites after correcting for the natural abundance of i8O. The evacuation/regassing procedure does not substantially alter the rate of phenol or B[u]P metabolism, since the rates of metabolism were 8590% of that obtained in open vessels (8). RESULTS AND DISCUSSION

Chemical

Characterization

The organic soluble metabolites of 3-hydroxy-@a]P formed in the presence of liver microsomes from phenobarbitaltreated rats, NADPH, and atmospheric oxygen can be separated and analyzed using reverse-phase HPLC with a methanolwater gradient (Fig. 1). Two major metabolites were noted with retention times of 0.59 and 0.84 relative to the 3-phenol (Table I). When liver microsomes from control, phenobarbital-, or 5,6-benzoflavone-treated rats were utilized, no differ-

138

PROUGH, SAEKI, AND CAPDEVILA

IOOXMeOH,~-.---~-.

I

I _._.-.-.-.

/./-i60%M&

A

60--a

~

A+

I 0

5

IO Time

15 (minutes)

20

25

30

FIG. 1. Chromatographic profile of 3-hydroxybenzo[a]pyrene and its metabolites. A 15-min incubation mixture containing the I-phenol, liver microsomal fractions from phenobarbital-treated rats, NADPH, and oxygen was extracted with ethyl acetate and dried under a stream of nitrogen. The residue was dissolved in methanol and analyzed using a reverse-phase HPLC column as described under Materials and Methods. The phenol was eluted by 100% methanol after 30 min. The dashed line indicates the change in the ordinate from 0.05 to 2.0 AUFS for the output of the detector at 254 nm. When 3-hydroxy-[7,10-‘4ClB[alP was used as substrate, 93-9756 of the added radioactivity was recovered in the ethyl acetate phase and the recovery of radioactivity from HPLC analysis was >94%.

ences in the metabolite pattern were noted. The less polar metabolite of the 3-phenol had absorbance and fluorescence emission spectra as well as HPLC retention time which were identical to those of authentic 3,6-B[u]P quinone. The more polar metabolite (A) (Fig. 1) had a HPLC retention time which was intermediate between those noted for the B[a]P dihydrodiols and the quinones. The absorbance maxima was similar to those of the B[u]P phenols, i.e., the major long wavelength absorbance maxima were noted to be between 400 and 450 nm. Both the absorbance and fluorescence emission maxima exhibited reversible shifts toward longer wavelengths upon the addition of base suggesting that metabolite A of the 3-phenol may be a phenolic compound. Mass spectral analysis indicated that one extra oxygen was incorporated into the molecule upon metabolism’(Table I). When similar experiments were performed using 9-hydroxy-qu]P, a much more complicated situation occurred. As seen in Fig. 2, only one major metabolite was formed which is identical to the more polar product (A) of the 3-phenol (Table I). Upon animal pretreatment with 5,6-

benzoflavone or phenobarbital two and three major metabolites were noted, respectively. The physical characteristics of the various metabolites of the 3- and 9phenol of @u]P are shown in Table I. It can be seen that the polar metabolite (A) from 3-hydroxy-B[u]P and the three metabolites from 9-hydroxy-B[u]P have a number of properties in common. They are as follow (Table I): (a) HPLC retention times intermediate between those of the B[u]P dihydrodiols and phenols; (b) absorbance maxima in the absence and presence of alkali similar to the B[u]P phenols; (c) emission fluorescence spectra in the absence and presence of alkali similar to the B[u]P phenols; and (d) the incorporation of one extra oxygen atom into their structure relative to the B[u]P phenols. The polar metabolites of the 3- and 9phenol (A) are identical in physical properties. The metabolite exhibits a shift in its wavelength maxima upon adding alkali (Fig. 3) which can be reversed if the sample is immediately neutralized. However, in alkaline conditions the phenolate spectrum changes with time to yield a broad absorbance maximum similar to those of the benzo[u]pyrene quinones. Finally, the

’ The retention times are expressed min). *The absorbance wavelength noted ‘The fluorescence wavelength noted 270 nm. ‘The values in parentheses are the ‘The values in parentheses are the ‘The values in parentheses are the ” Metabolite forms a quinone upon

3-Hydroxy 9-Hydroxy 3,fGQuinone 4,5-Dihydrodiol Peak A (g-phenol) Peak B (g-phenol) Peak C (g-phenol) Peak A (3-phenol) Peak Q (3-phenol)

Compound 424 418 480 322 435 427 424 435 480

(458) (442) (480) (322) (444, (420, (419, (444, (480)

431 422 442, 375 442 436 432, 442 442,

268 268 282 286 284 284 284 284 282

of the compounds in 50% methanol in water. maximum of the @a]P derivatives in 80% methanol.

and a methanol-water

METABOLITES

column

(520)f (513) 570 (570) (375) (533) (532) 454 (525) (533) 570 (570)

Emission maximum” (nm)

AND THE PHENOL

a nBondapak-C18

1

relative intensities of the major fragments measured by mass spectrometry. wavelength maxima in the presence of 0.1 N KOH in 50% methanol. maxima in the presence of 0.15% triethylamine in 80% methanol. standing in alkaline solution.

wavelength maximum emission wavelength

using

STANDARDS

473y 458)y 461) 473)61

B[a]P

Absorbance maximum* (m-4

OF VARIOUS

to 3-hydroxybenzo[a]pyrene

is the longest is the major

relative

1.06 0.96 0.83 0.48 0.59 0.68 0.78 0.59 0.84

Retention time”

PROPERTIES

TABLE

The

samples

gradient

were

(60-100%

excited

over

at

40

(lOO), 239 (53), 134 (41), 119 (93) (lOO), 239 (49), 134 (67), 119 (88) (lOO), 254 (31), 226 (51), 224 (29) (lOO), 268 (66), 255 (13), 239 (47) (XXI), 268 (9), 255 (23), 239 (6) (38), 268 (lOO), 255 (98), 239 (27) (12), 268 (loo), 255 (5), 239 (52) (lOO), 268 (12), 255 (31), 239 (8) (lOO), 252 (28), 226 (61), 224 (34)

m/cd

140

PROUGH, SAEKI, AND CAPDEVILA

6

5

IO

I5

Time

(minutea)

2’0

$5

30

FIG. 2. Effect of animal pretreatment regimen on the chromatographic profile of 9-hydroxy-qu]P and its metabolites. Samples were analyzed as described in Fig. 1. The dashed line indicates the change in the ordinate from 0.2 to 2.0 AUF for the output of the detector at 254 nm. When 9hydroxy-[7,10-‘“Cma]P was used as substrate, 95-97% of the added radioactivity was recovered in the ethyl acetate phase and the recovery of radioactivity from HPLC analysis was >95%.

mass spectra of the metabolites show a fragmentation pattern which can be described by the following molecular ion and its fragments: M, M-16, M-29, and M-45. Clearly, one additional oxygen atom is added to the phenol to form the metabo-

lite. However, the mass spectral fragmentation pattern is similar to the B[a]P epoxide (11) in that the first fragment formed is M-16 (the loss of oxygen). The remaining fragments are similar to those formed by the @alp phenols. While there remains

0.16. w Lj 4 m :

0.12.

0.08-

,” 4 0.04-

01

300

400 WAVELENGTH,

500

600

1 nm 1

FIG. 3. Absorbance spectra of metabolite A from I)-hydroxy-@ujP. Metabolite A was dissolved ). The solution was made 0.1 N in KOH in 50% methanol in water and the spectrum recorded (and the spectra recorded within 3 min (0 0) or after 30 min (- - -).

BENZO[a)PYRENE

PHENOL

141

METABOLISM

0.16.

: fi 2 $ m Q

0.12.

o.os-

0.04

-

O-

300

400 WAVELENGTH,

500

600

(nm)

FIG. 4. Absorbance spectra of metabolite B from 9-hydroxy-Mu]P. Metabolite B was dissolved in 59% methanol in water and the spectrum recorded (-). The solution was made 0.1 N in KOH and the spectra were recorded within 3 min (0 0) or after 39 min (- - -).

rapidly rearrange to form phenols (12). These properties would agree with the fact that a metabolite of the g-phenol binds to DNA and protein (5-7). The mass spectra of the metabolites have some differences. As reported by McCaustland et UI!. (ll), the fragmentation patterns for the hydroxy-B[a]P derivatives consist of an intense M-29 peak with a

some question as to the structure, the absorbance and fluorescence changes on addition of base or upon reacidification are similar to that obtained with the B[a]P phenols. While we assume that these metabolites are dihydroxy derivatives of B[u]P, it must be remembered that small amounts of epoxide may be present since it is known that these epoxy derivatives

100%

0

IO

20 TIME

30

40

(minutes)

FIG. 5. Chromatographic profile of metabolite B before and after incubation in 0.1 N KOH. A solution of metabolite B identical to that shown in Fig. 4 was made 0.1 N in KOH and aliquots were neutralized at a given time prior to extraction. The samples were extracted with ethyl acetate, the organic phase evaporated to dryness, and the residues analyzed by HPLC with a 40-min gradient from 39 to 199% methanol. The samples were neutralized either immediately after addition of KOH (-) or 39 min after addition of KOH (- - -).

PROUGH,

SAEKI,

AND

CAPDEVILA

0.16.

: 5 z z

0.12-

0.08-

z 0.04.

O-

300

400 WAVELENGTH,

500

600

(nm)

FIG. 6. Absorbance spectra of metabolite C from 9-hydroxy-qu]P. Metabolite C was dissolved in 50% methanol in water and the spectrum recorded (-). The solution was made 0.1 N in KOH and the spectrum recorded after 30 min (0 0).

measurable M-28 peak. The arene oxides behave similarly, but have a small M-16 ion which has been suggested to be unique for arene oxides. In addition, Jerina et al. (13) have reported the ultraviolet and visible absorbance spectra of all 12 B[u]P phenols and McCaustland et al. (11) have documented the uv-visible spectra of all of the major metabolites of B[u]P. The phenols all have intense absorbance transitions with maxima which extend into the region from 390-440 nm. With the exception of the 7&oxide- and ‘7,Sdihydrodiol of B[a]P, which absorb at approximately 370 nm and lower, all of the other B[u]P oxides and dihydrodiols have absorbance bands which lie below 340 nm (11, 13). Based on these observations, we interpret the data of Table I and Fig. 3 to indicate that metabolite A of the 3- and 9phenol most likely is the 3,9-dihydroxy derivative. This conclusion is based on the low amount of M-16 ion and the pronounced M-29 ion obtained by mass spectral analysis. The compound undergoes a bathochromic shift on addition of base similar to other B[u]P phenols (Fig. 3) and is eventually oxidized chemically to a quinone. Metabolite B of the g-phenol behaves like a phenol in alkaline solution (Fig. 4), but has an intense M-16 ion suggestive of

a putative phenol-epoxide. In addition, metabolite B is much more rapidly dized to a quinone (Figs. 4, 5) than other metabolites. This may suggest TABLE OXYGEN-H

Substrate

II

ENRICHMENT OF B[a]P METABOLITES FORMED IN THE PRESENCE OF I80 MOLECULAR OXYGEN

Metabolite”

Percentage ‘80 enrichmentb

@alp

3-Hydroxy-B[a]P B[a]P 4,5-dihydrodiol

98 48

3-Hydroxy-

Metabolite A Ma]P 3,6-quinone

84 60’

B[alP

the oxithe that

” Metabolites were purified by reverse-phase HPLC using a pBondapak Cl* column and rechromatographed by normal-phase HPLC using a PBondapak NH2 column. bMass spectra were obtained using direct probe analysis. The percentage “0 enrichment was determined by monitoring the M and M + 2 parent ion peaks for the various metabolites. ’ When the B[u]P 3,6-quinone was incubated with H2 la0 for 5 min in the presence of liver microsomal protein, extracted, and purified by HPLC, a 10-15s incorporation of “0 from Hz I80 was detected indicative of a slow exchange with water.

BENZO[a]PYRENE

PHENOL TABLE

143

METABOLISM

III

ENZYMATIC CHARACTERIZATION OF THE PHENOL MONOOXYGENASE ACTIVITY OF RAT LIVER MICROSOMAL FRACTIONS Spectrophotometric analysis Pretreatment regimen

K, (PM)

V

Chromatographic A

B

(nmol/min/mg)

analysis

C 3,6-Quinone (nmol/min/mg)

Total

3-Hydroxybenzo[a]pyrene” 125 42 11

Control Phenobarbital 5,GBenzoflavone

3.0 8.0 2.0

1.4 1.6 1.9

-6 -

-

0.05 0.62 0.25 0.36

0.38 -

2.7 5.1 3.4

4.1 6.7 5.3

-

0.26 1.27 0.67 0.80

9-Hydroxybenzo[a]pyrene’ Control Phenobarbital 5,6-Benzoflavone 3-Methylcholanthrene

50 9 19 -

0.25 2.0 1.7 -

0.21 0.26 0.41 0.44

’ The maximal rate of metabolism was measured spectrophotometrically COefficient

Of 21,000

Me’

at 448 nm assuming an extinction

Cm-‘.

” Not detected. ‘The maximal rate of metabolism was measured spectrophotometrically tabolite A has an extinction coefficient of 26,000 Mm’ cm-‘.

the second hydroxyl is located close to or on the benzo-ring allowing a more facile oxidation of the dihydroxy compound to a quinone. Production of metabolite C from the g-phenol is effectively induced by animal pretreatment with phenobarbital; this situation is similar to the induction pattern of the 4,5-oxide and 4,5-dihydrodiol of B[a]P. It also shows a phenolate ion spectra (Fig. 6), but is not easily oxidized to a quinone-like compound. Metabolite C also has an intense M-16 ion suggestive of a phenol-oxide intermediate consistent with the suggestion of King et al. that a putative 9-hydroxy-4,5-oxide metabolite may exist (12). The products isolated may be dihydroxy derivatives which fragment to M-16, as well as M-29. Contamination of the sample by the g-phenol is unlikely since two HPLC column conditions with widely differing retention times were used during the purification of the samples. Experiments were performed using an atmosphere of ‘*02 to determine whether these metabolites were formed by a mech-

at 436 nm assuming that me-

anism similar to the other hydroxylated products obtained by the cytochrome P450-dependent monooxygenase of mammalian liver. Table II shows the incorporation of ‘*O into the 3-phenol and 4,5-dihydrodiol of B[a]P. As reported by Yang et al. (14), one atom of atmospheric oxygen is incorporated into the products of benzo[alpyrene as predicted by the work of Hayaishi et al. (15) and Mason et al. (16). There was also one atom of atmospheric oxygen incorporated into the two products of 3-hydroxybenzdalpyrene demonstrating that the same stoichiometry of oxygen incorporation exists for the products of the 3-phenol. The lower incorporation into the 3,6-quinone and metabolite A could be due to either a small contamination with 1602, to an exchange with water from the incubation mixture, or to minor differences in “0 incorporation. We have observed a 10-15s exchange of the oxygen of the 3,6quinone under these conditions over a 5min period (Capdevila, Saeki, and Prough, unpublished results).

144

PROUGH,

SAEKI, TABLE

AND

CAPDEVILA

IV

THE EFFECT OF INHIBITORS ON THE PHENOL MONOOXYGENASE ACTIVITY OF LIVER MICROSOMES Percentage inhibition” Addition

3-Phenol 3-Phenol 3-Phenol 3-Phenol 3-Phenol 3-Phenol

- NADPH

98

-Q

- Oxygen - Microsomal protein 7,8-BF (60 /.JM) TCPO (0.2 mM) co:o* (4:1, v/v)

86 99 42(45) 5 32

-

-NADPH

95

-

- Oxygen - Microsomal protein 7,8-BF (60 PM) TCPO (0.2 mM) co:02 (4:1, v/v)

87 97 65(68)

-69) 5W-Y 48

g-Phenol g-Phenol g-Phenol g-Phenol O-Phenol O-Phenol ’ The concentration of ml protein from PB- or repetitive scan difference for %-phenol metabolism for g-phenol metabolism b Not determined.

or deletion

5,6BF

Substrate

PB

WY 38

-(52) 10 40

aromatic phenols used was 100 PM and the reaction was run at 37°C with 0.5 mg/ 5,6-BF-treated rats. The values were obtained by monitoring the reaction using spectral analysis; those in parentheses were obtained by HPLC analysis. The values by PB and 5,6-BF microsomal protein were 7.8 and 2.2 nmol/min/mg. The values by PB and 5,6-BF microsomal protein were 2.1 and 1.6 nmol/min/mg.

Enzymatic Characterization Metabolism

of Phenol

The characterization of the metabolism of B[a]P phenols was performed using repetitive scan spectrophotometric analysis (Table III). The apparent Michaelis-Menton constants were determined using repetitive scan spectrophotometry (8) by measuring the change in absorbance at 448 nm assuming that the principal product of 3-phenol metabolism is the 3,gquinone which exists as the hydroquinone during the steady state of the reaction (17) or by measuring the formation of the metabolite A, the major metabolite of the g-phenol at 436 nm. The K, values obtained for the two phenols ranged from 10 to 125 pM. Microsomes from livers of 5,6-BF- or PBpretreated rats had lower K, values than the controls; a situation similar to that seen for the K, of the parent hydrocarbon benzo[a]pyrene (18). Table III also shows the effect of animal pretreatment on the rates of product formation from the 3- or g-phenol of Ha]P measured by HPLC analysis coupled with

liquid scintillation techniques. The rates of metabolism were determined by the two methods (spectrophotometry and HPLC) and they gave good agreement except when liver microsomes from 5,6-benzoflavone-treated rats were used. For 3-phenol metabolism, the spectrophotometric technique is dependent upon the NADPH-quinone reductase activity of liver microsomes (1’7); the reductase activity may not have been sufficient to fully reduce the quinone to its hydroquinone or metabolite A was formed in large quantities relative to the hydroquinone measured. Animal pretreatment had a pronounced effect on metabolism of the two phenols by rat liver microsomes. For the 3-phenol, metabolite A formation was not enhanced, but 3,6quinone formation was increased 88 and 26% by PB and BF, respectively. Animal pretreatment with phenobarbital had little or no effect on the formation of metabolite A from the g-phenol, but greatly increased the rate of formation of metabolites B and C. Pretreatment of rats with and 3-methylcholan5,6-benzoflavone threne increased the rate of formation of

BENZO[a]PYRENE

400

450 Wavelenglh

500

550

PHENOL

I

(nm)

FIG. 7. Spectral analysis of the metabolism of 3and P-hydroxy-B(a]P. Absolute spectra of reaction mixtures containing (A) 9-hydroxy-B[u]P and (B) 3hydroxy-B(a]P were recorded either prior to addition of NADPH (--), at 5 min during the steady state of metabolism (. ) or after all of the NADPH was depleted at 20 min by metabolism (- - -). The sample and reference cuvettes contained 0.5 mg/ml of microsomal protein from PB-treated rats. NADH (209 PM) was added to both the reference and sample cuvette to eliminate the contribution of cytochrome bs to the spectra. No NADPH regenerating system was included and the NADPH concentration was 100 PM.

metabolite A of the g-phenol by loo%, but increased formation of metabolite B by 500-700% (no metabolite C was noted). The effects of various cytochrome P-450 inhibitors on the metabolism of the two phenols are shown in Table IV. It can be seen that the reactions require NADPH, OZ, and microsomal protein. In addition, a carbon monoxide/oxygen atmosphere (4:1, v:v) and 7,Sbenzoflavone were effective inhibitors in the same manner as shown by Wiebel (19). Trichloropropene2,3-oxide (0.2 mM), the inhibitor of epoxide hydratase, had no effect on metabolism indicating that if epoxides are formed, they are either too unstable to be metabolized by the epoxide hydrolase or are not

METABOLISM

145

substrates for the enzyme. This result agrees with the lack of effect of this agent on the covalent binding to DNA or bacterial mutagenesis by the metabolic products of 9-hydroxy-@a]P (7). Taken together, the data on the effect of animal pretreatment regime and of inhibitors clearly indicate that the phenol monooxygenase of rat liver microsomes is a cytochrome P-450-dependent enzyme system. During the steady state of metabolism, one can use repetitive scan difference spectrophotometry to evaluate the relative proportion of dihydroxy and quinone products (8, 10). As can be seen in Fig. 7, there was no appreciable absorbance in the region above 455 nm during the steady state of metabolism of either phenol. However, when limiting amounts of NADPH were added, measurable absorbance is noted above 455 nm after all of the NADPH had been utilized. Clearly, the NADPHquinone reductase activity of liver microsomes, which is apparently due to the presence of NADPH-cytochrome P-450 reductase, maintained the metabolites in a highly reduced state in the presence of NADPH. This is particularly noticeable for the metabolites of the 3-phenol since appreciable amounts of the B[u]P-3,6-quinone are formed. Two primary biological end points can be used to study the metabolic activation of benzo[a]pyrene and its metabolites. These are the induction of mutations in procaryotes (20) or eucaryotes (3) and the activation to metabolites which can alkylate DNA (21). Recently we reported the close correlation between the microsomemediated mutagenesis and DNA alkylation assays for B[a]P and its g-phenol (7). The g-phenol of B[a]P is a better “premutagen” and “prealkylating” agent than either B[a]P or the 3-phenol of fla]P. The g-phenol does cause significant epidermal hyperplasia upon skin painting (22) and does appear to be an immediate precursor to the primary DNA adduct formed in hepatocytes treated with B[a]P (23). Recently, Sims and Grover have reported that the DNA adducts of the phenol oxides can be detected in rodent skin using flu-

146

PROUGH,

SAEKI,

orescence techniques (24); this observation demonstrates the importance of the phenol metabolites in tivo. In addition, Shen et al (25) have shown that the quinone products of the phenols are potent inhibitors of the reaction which converts the 7,8-dihydrodiol to the 7,8-dihydrodiol-9,10-oxide of B[a]P. While the role of the “bay region diolepoxides” of B[u]P and several other polycyclic hydrocarbons as powerful mutagens and complete carcinogens is well established, the process of tumor induction in tivo could involve interaction of multiple reactive chemical species with their target molecules. This biological process may be complicated by the diverse number of metabolic products, their chemical reactivity, and the complement of enzymes involved in their formation, stabilization, and disposition. Since all of these factors are at play in tumor production, the exact biological implication of our findings on phenol metabolism, i.e., the formation of “phenol epoxides,” the apparent reactivity toward protein and DNA (‘7), the possible role of redox cycles in generating semiquinone, hydroquinone, or reactive species of oxygen (17), and the effect of phenol metabolites on diol-epoxide formation (25) are difficult to assess at this time. ACKNOWLEDGMENTS The authors express their gratitude to Ms. V. W. Patrizi for her expert technical assistance. This work was supported, in part, by USPHS Grant HL 19654 and NC1 Contract NOl-CP-33362. RAP is USPHS Research Career Development Awardee HL 00255. REFERENCES 1. SIMS, P., GROVER, P. L., SWAISLAND, A., PAL, K., AND HEWER, A. (1974) Nature &mn%m) 252, 326-328. 2. SELKIRK, J. K., CROY, R. G., AND GELBOIN, H. V. (1974) Science 184, 169-171. 3. HUBERMAN, E., SACHS, L., YANG, S. K., AND GELBOIN, H. V. (1976) Proc Nat. Sti USA 73,607611. 4. THAKKER, D. R., YAGI, H., Lu, A. Y. H., LEVIN, W., CONNEY, A. H., AND JERINA, D. M. (1976)

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