Differences in the mechanism of NADPH- and cumene hydroperoxide-supported reactions of cytochrome P-450

ARCHIVES OF BIOCHEMISTRYAND BIOPHYSICS Vol. 200, No. 1, March, pp. 186-195, 1980

Differences

in the Mechanism of NADPH- and Cumene HydroperoxideSupported Reactions of Cytochrome P-4501 J. CAPDEVILA,

Department

of Biochemistry,

R. W. ESTABROOK, AND R. A. PROUGH2

The University

of Texas Health Science Center at Dallas,

Dallas,

Texas %.235

Received August 21, 1979; revised October 23, 1979 The mechanism of NADPH- and cumene hydroperoxide-supported hydroxylation of benzo(a)pyrene as catalyzed by liver microsomes was studied using high pressure liquid chromatography, fluorescence, and spectrophotometric methods. Repetitive scan difference spectral analysis clearly demonstrated that during the steady state of these reactions, different products were formed. While the major products noted with NADPH were phenols, only low concentrations of phenols were observed in the presence of cumene hydroperoxide using all three analytical methods. With the organic hydroperoxide, the metabolite profile was shifted from the preponderate production of phenols and dihydrodiols to the production of the three quinone isomers of the hydrocarbon. At several concentrations of cumene hydroperoxide, epoxide hydrase activity and the stability of the arene oxide substrates tested were unaffected during a 2-min incubation period. The transient nature of benzo(a)pyrene phenol formation was investigated; the 3- and g-phenols were easily oxidized to quinones by cumene hydroperoxide in a cytochrome P-450-dependent oxidation process which most likely involves the formation of free radicals by a one-electron process. These results indicate that the reaction mechanism operative in the presence of the organic hydroperoxide differs in several regards from that functional in the presence of NADPH and that a common oxidative mechanism may not exist.

During the NADPH-supported, liver microsome-catalyzed oxidation of benzo(a)pyrene (B(u>P>,~ two general classes of metabolites are formed: The first class consists of dihydrodiols and certain phenols (e.g., 9-hydroxy-B(a)P) which are formed by epoxidation followed by hydration and/or rearrangement (1, 2). The second class consists of the 6-phenol, possibly portions of the 3-phenol, and the quinones of B(a)P which are formed by yet unidentified mechanisms (3-5). It has been suggested

that the 3- and 6-phenol metabolites might arise from either direct insertion of an oxygen atom across carbon-hydrogen bonds or from the rapid rearrangement of unstable epoxide intermediates (2). However, Nagata et al. (3) and Lorentzen et al. (4) have provided considerable evidence that the quinones most likely are formed by oneelectron oxidation reactions involving 0x0 radicals as intermediates. The metabolites of benzo(u)pyrene can be monitored with accuracy using a number of analytical methods: absorbance spectroscopy (6), fluo1 This work was supported in part by NC1 Contract rescence spectroscopy (‘7,.S), and thin layer NO1 CP 33362 (RAP/RWE), NIH Grants HL 19654(RAP) and GM 16488(RWE), and R. A. Welch (9) or high pressure liquid chromatography Foundation Grant I-616(RAP). R. A. P. is a Research (l,lO). The availability of these methods has Career Development Awardee, HL 00255. A pre- allowed for the detailed study of the liminary report of this work was presented at the metabolite profile for benzo(u)pyrene and 63rd Annual Meeting of the FASEB, Dallas, Texas, has provided the chemical techniques April l-10, 1979, Abstract 1568. necessary to search for proximal carcino* To whom correspondence should be directed. gens, such as the B(u)P diol epoxides 3 Abbreviations used: BF, 5,6-benzoflavone; B(a)P, (1 l- 13). In general, other substrates for the benzo(a)pyrene; CuOOH, cumene hydroperoxide; cytochrome P-450-dependent monooxygenhplc, high performance liquid chromatography; PB, ase do not yield the number of products seen phenobarbital. 0003-9861/80/030186-10$02.00/O Copyright 0 1980by AcademicPress, Inc. All rights of reproductionin any form reserved.

186

NADPH- AND CUMENE HYDROPEROXIDE-SUPPORTED

in the case of B(a)P. The diverse structures of these metabolites and the availability of suitable analytical methods allow one to probe the mechanism of hydroxylation of the polycyclic aromatic hydrocarbon in a manner not possible with most other cytochrome P450-dependent monooxygenase substrates. Hrycay and O’Brien (14, 15) have shown that cytochrome P-450 can act as a peroxidase since the oxidation of electron donors such as NADH, NADPH, and other reductants occurs in the presence of organic hydroperoxides. Cumene hydroperoxide and other organic peroxides have been used as model reagents for the study of peroxidative metabolism catalyzed by cytochrome P-450 and other hemoproteins (16-20). It has been shown that cytochrome P-450 serves as a unique catalyst in reaction mixtures containing a number of organic and inorganic oxidants, such as sodium periodate, sodium chlorite, iodosobenzene, and hydrogen peroxide. This phenomenon applies to many of the cytochrome P-450dependent reactions and it was also noted that several alcohols could be oxidized to their respective aldehydes by cumene hydroperoxide and either catalase or cytochrome P-450 (21). A number of these reports have concluded that the organic peroxide-dependent reactions of the cytochrome serve as models for the NADPHdependent oxygen activation mechanism for the cytochrome P-450 and other hemerequiring monooxygenases. The present report provides evidence suggesting that there are significant differences between the two types of reactions with regard to the metabolism of benzo(a)pyrene and presumedly, the active oxygen species involved in the reactions catalyzed by liver microsomal fractions obtained from either phenobarbital or 5,6-benzoflavone pretreated rats. MATERIALS

AND METHODS

Microsomal suspensions were prepared as described previously (6) and the specific content of cytochrome P-450 was measured using the method of Omura and Sato (22). [7,10-‘4C]Benzo(a)pyrene (specific radioactivity,21.7 mCi/mmol) was obtained from Amersham Inc. (Arlington Heights, Ill.), was diluted with

REACTIONS

187

unlabeled B(a)P to obtain a specific radioactivity of 2 mCilmmo1, and was purified by hplc (isocratic conditions, 80% methanol in water using a Waters’ Associates @Bondapak Cl8 column, Milford, Mass.). Cumene hydroperoxide was purchased from Matheson Coleman and Bell (Norwood, Ohio) and used without further purification. The pyridine nucleotides, sodium isocitrate, and isocitrate dehydrogenase (Type IV) were purchased from the Sigma Chemical Company (St. Louis, MO.). A standard reaction mixture containing 0.5 mgiml liver microsomal protein, 5 mM MgCIZ, 150 mM KCl, and 50 mM Tris-HCl buffer, pH 7.5 was preincubated at 37°C for 3 min. Fifty microliters of B(a)P in acetone was added to the incubation mixture (80 pM final concentration) prior to initiation of the reaction with cumene hydroperoxide (12-120 pM final concentration). The NADPH-dependent hydroxylation of B(a)P was performed using a similar incubation mixture, but which included 2 mM sodium isocitrate, 1 IUiml isocitrate dehydrogenase, 1 pM rotenone, 200 pM NADH, and 400 pM NADPH. The addition of rotenone and NADH had no measurable effect on metabolism ((6), unpublished results), but eliminated the spectral contribution of cytochrome b5 during the spectrophotometric analysis. Repetitive scan spectrophotometric analyses of the metabolic products were performed as previously described by Prough et al. (6). The hplc analysis of metabolic products were performed as described by Prough et al. (6) with some modification. The samples were analyzed using a reverse phase hplc column (PBondapak C,,) and a 60 to 100% methanol-water linear gradient (1% change/ min) at a flow rate of 1.3 ml/min. The separation was monitored by 254 nm absorbance and samples were collected every 30 s for radioactive analysis. The recovery of radioactivity from the hplc column ranged from 94-103%. When ethyl acetate was used to extract the B(a)P and its metabolites, the recovery of radioactivity from the aqueous phase was >99% in all cases. The fluorescence assay, a modification of the method of Dehnen et al. (8), was performed by termination of the reaction with a half-volume of chilled acetone, centrifugation at low speed, and addition of an aliquot of the supernatant to a solution of 7.5% triethylamine in water. The fluorescence of the phenols was measured using an excitation wavelength of 467 nm and emission wavelength of 518 nm. Epoxide hydrase activity was measured using the method of Jerina et al. (23); the specific radioactivity of [3-3H]phenanthrene-9, lo-oxide, [6-3H]benzo(a)pyrene-7,8-oxide, and [11,12-3H]benzo(a)pyrene-11,12-oxide was identical to that reported by Jerina et al. (23). The reaction mixture was preincubated for 5 min at 37°C and the hydrocarbon was added in acetonitrile to initiate the reaction. The reaction was terminated after 2 min by the addition of chilled tetrahydrofuran and the dihydrodiols were

188

CAPDEVILA,

ESTABROOK, AND PROUGH

quantitated using duplicate samples as previously described (23). The NADPH- or cumene hydroperoxide-dependent generation of free radicals from B(a)P or B(u)P phenols was analyzed using the benzene extracts of 25ml reaction mixtures (described above) containing either B(a)P (80 PM), 3-phenol (100 PM), or &phenol (100 PM). The benzene extracts were evaporated under vacuum using a rotary evaporator and were dissolved in 0.3-0.5 ml of dry benzene. The sample was transferred into quartz ESR tubes and bubbled with oxygen-free argon for 15 min. Electron spin resonance spectra were recorded using a Varian Model E-4 ESR spectrometer at room temperature. A PDP-11 Minicomputer (Digital Equipment Corporation) was used to collect and process the data. Authentic 60x0 radical was generated by shaking 5 ml of 1 mM potassium ferricyanide and 5 ml of benzene containing the 6-phenol, removing the benzene phase, and preparing the sample for ESR measurement as described above. RESULTS

Repetitive scan difference spectrophotometric analysis of aerobic incubation mixtures containing B( a)P, liver microsomes from phenobarbital-treated rats, and either cumene hydroperoxide or NADPH revealed considerable differences between the two reactions (Fig. 1). The cumene hydroperoxide-supported reaction exhibited the time-dependent formation of a broad absorbance band at around 460 to 470 nm which indicates the presence of B(a)P quinones. A small, but transient absorbance change existed at approximately 400 nm probably due to the formation of either the 3- or 6-phenol of B( a>P. The interpretation of the absorbance changes which occur between 380 and 440 nm is complicated by the possible contribution in that region of spectral changes associated with the interaction of eumene hydroperoxide with cytochrome P-450 (24). Under the conditions of our study, the contribution of processes involving cumene hydroperoxide alone amounted to less than 20% of the total absorbance change reported here (Capdevila and Prough, unpublished results). In addition, the maxima and shape of the spectra were markedly different from those due to the cumene hydroperoxide interaction. During the NADPH-dependent metabolism of B(a)P, there was no significant

NACW

T

004A

1

FIG. 1. Difference spectral analyses of the metabolism of benzo(a)pyrene supported by cumene hydroperoxide or NADPH. A standard reaction mixture containing B(a)P and liver microsomes from phenobarbital-treated rats (see Materials and Methods) was divided into cuvettes and placed in the sample and reference compartment of an Aminco DW-2 spectrophotometer in the split beam mode thermostated at 37°C. After a baseline of equal light absorbance was established, the reaction was initiated with either NADPH (400 pM, final concentration) or cumene hydroperoxide (70 pM, final concentration). The time-dependent changes in absorbance between 370 and 670 nm were recorded by repetitive scanning at 10 nmis.

increase in absorbance beyond 440 nm suggesting that the concentration of quinones which may exist during the steadystate of the reaction is very low due to the presence of an active microsomal quinone reductase (25). The shoulder at approximately 440 nm most probably is due to the presence of benzo( u)pyrene hydroquinones since upon depletion of NADPH in the reaction mixture, the 440-cm shoulder decreased with a concomitant increase in absorbance at 480 nm indicative of B(u)P quinones (data not shown). The presence of phenols was indicated by the increased absorbance as a function of time at 401,409, and 428 nm corresponding to the absorbance maxima of the 3-phenol, g-phenol, and a mixture of both, respectively (6). The contribution of an oxygenated form of reduced cytochrome P-450 (26) to the

NADPH- AND CUMENE HYDROPEROXIDE-SUPPORTED

spectra was most probably negligible since there was not an instantaneous increase in absorbance in the 440- to 490-nm region as is noted with hexobarbital and the amount of absorbance expected for such a complex under our experimental conditions would be less than 20% of that actually noted in Fig. 1. High performance liquid chromatography analyses of incubation mixtures, identical to those used in the spectrophotometric study, corroborated the interpretation of the spectral changes suggesting different patterns of product formation. The NADPHsupported reaction gave the typical hplc profile of the organic soluble metabolites formed during the first minute of metabolism (Fig. 2). However, the profile obtained when cumene hydroperoxide was used as the oxidant is clearly different. The product distribution indicated that the principal

REACTIONS

reaction in the presence of the organic peroxide is switched to the production of quinones at the expense of dihydrodiol and phenol metabolites. In Table I, the relative proportions of the organic soluble metabolites produced by liver microsomes from PB- or BF-pretreated rats with either NADPH or cumene hydroperoxide are contrasted. When B(a)P was metabolized in the presence of NADPH, liver microsomes from 5,6-benzoflavonetreated rats were more active than those obtained from phenobarbital-treated rats. The opposite occurred when cumene hydroperoxide was added to the reaction mixture. A similar result has been noted for the aromatic hydroxylation of acetanilide (5) and benzo(a)pyrene (27) in the presence of the organic hydroperoxide. A large percentage of the NADPH-dependent prod-

CIJOOH /I OOIA-

9JO I

45 7.0 I I

wit I

189

652 I

9 I

FIG. 2. The hplc separation of benzo(a)pyrene metabolites produced by the cumene hydroperoxideor NADPH-supported reaction. The retention times of the authentic standards for the three dihydrodiols, three quinones, two phenols, and B(a)P, respectively, are indicated in the figure. In order to contrast the metabolite profiles of the cumene hydroperoxide and NADPH-dependent reactions, the sample injected onto the hplc column for the NADPH sample contained approximately fivefold more total product than that for cumene hydroperoxide baaed on time, aliquot size, and total protein (see Materials and Methods).

190

CAPDEVILA,

ESTABROOK, AND PROUCH TABLE I

NADPH- AND CUMENE HYDROPEROXIDE-DEPENDENT

METABOLISM OF BENZ~&)PYRENE

Percentage of total metabolitesa

Addition

Total metabolism (nmoYmin/mg)

NADPH CuOOH

5.4b IT 0.6 5.7 2 0.4

NADPH CuOOH

5.9 f 0.4 2.0 f 0.2

Dials 1

2

Quinones 3

1

Phenols

2

3

Liver microsomes from PB-treated rats 9.2 f 1.0 9.4 -c 1.0 3.9 + 0.4 9.0 + 0.9 10.8 2 1.2 7.8 + 0.9 1.5 + 0.2 4.8 -+ 1.3 0.1 + 0.4 43.0 + 3.8 38.0 r 4.5 13.0 t 0.3

1

2

2.8 +- 0.3 4’7.1 t 4.2 0.4 k 0.3 0.0

Liver microsomes from BF-treated rata 11.6 f 1.0 5.5 f 0.5 8.0 k 0.9 6.5 t 0.7 6.7 k 0.7 7.4 -c 0.8 13.1 2 1.2 41.2 + 4.0 5.3 f 0.7 1.0 f 2.0 0.2 -c 0.2 31.0 -c 1.0 34.0 ? 2.6 10.6 k 3.0 4.1 ?I 1.1 13.5 + 1.8

a The metabolites shown represent the amount ofradioactive product which comigrated with the following B(a)P metaholites: 9,lOdihydrodiol, diol 1; 4,5dihydrodiol, diol 2; ‘IJ-dihydrodiol, diol 3; l,Bquinone, quinone 1; 3,6quinone, quinone 2; 6,12 quinone, quinone 3; 9-phenol, phenol 1; and 3-phenol, phenol 2. The recovery of radioactive products and substrate into the ethyl acetate phase from reaction mixtures containing either NADPH or cumene hydroperoxide was greater than 99% and recovery from the hplc was greater than 94% in all csses. b The values of the percentage of total metabolites are given with the standard deviation of the mean (n = 4 in all cases).

ucts is formed by epoxidation followed by hydration and/or rearrangement relative to the products obtained with cumene hydroperoxide. This can be noted most simply by comparing the percentage of metabolites formed as quinones relative to dihydrodiols. In the cumene hydroperoxide-supported reaction, high concentrations of the three quinone isomers were produced at the expense of dihydrodiols and phenols. In the case of liver microsomes from PB-treated rats, 94% of the total B(a)P metabolites formed in the presence of cumene hydroperoxide were quinones. Similar results were obtained with microsomes from BFtreated rats (Table I). Although the hplc system we have used does not allow one to distinguish between the 3- and 6-hydroxy-B(a)P, analysis of the quinone fraction showed that significantly higher amounts of the 1,6- and 3,6-quinones were produced relative to the 6,12-quinone indicating that both phenols may be formed as intermediates in either reaction condition. The chemical oxidation of 6-hydroxyB(a)P led to the formation of nearly equimolar amounts of the three different quinone isomers ((4); Capdevila and Prough, unpublished results). The relatively small amounts of B( a)P-phenols recovered during the analysis of the cumene hydroperoxidedependent reaction suggest that the phenols

may be rapidly further oxidized. In order to test this hypothesis, the stability of selected B(a)P metabolites in the presence or absence of 100 PM cumene hydroperoxide at 37°C was first tested by hplc analysis of the ethyl acetate extract of the reaction mixtures (Table II). During the first 2 min, the 9,10-, 4,5-, and 7,8dihydrodiols were stable in the presence of cumene hydroTABLE II STABILITY OF BENZO(U)PYRENE METABOLJTES”

Percentage loss of Metabolite Substrate

Enzymatic

Nonenzymatic

9,16-Diol 4,5Diol 7,8-Diol S-Phenol g-Phenol 6-Phenol

8 7 4 31 17 65

0 9 2 7 0 20

a The reactions were run for 2 min at 37°C with either 0.5 mg/ml native (enzymatic) or boiled (nonenzymatic) microsomal protein from the livers of PBtreated rats. The concentration of hydrocarbon used was 56 KM and the concentration of cumene hydroperoxide was 166 PM. The amount of conversion was determined by quantitation of the decrease of substrate concentration using the peak area of the 254nm trace from the hplc detector.

NADPH- AND CUMENE HYDROPEROXIDE-SUPPORTED

peroxide and either native or heat-denatured microsomes (>9’7% recovery of parent compound). In contrast, 45% of the 6-phenol, 24% of the 3-phenol, and 17% of the g-phenol were enzymatically oxidized within the first 2 min in the presence of intact microsomes from PB-treated animals and cumene hydroperoxide. The product of the oxidation of the 3-phenol was shown to be the 3,6-B(a)P quinone, but no ethyl acetate-soluble products were noted for the g-phenol upon hplc analysis. The organic peroxide caused significant conversion of the 6-phenol to yield the three quinone isomers. The transient nature of phenol formation was investigated by using the fluorescence assay for B(a)P phenols (8). When B(a)P hydroxylation is monitored by measuring the fluorescence of the phenols in alkaline media, one obtains an estimate of the amount of the 3- and g-phenols formed; all of the other B(a)P phenols, including the 6-phenol, have low fluorescence emission in alkali (28). As seen in Fig. 3, the concentration of cumene hydroperoxide had a pronounced effect on the amount of fluorescent product during the reaction. Microsomes prepared from PB-pretreated rats appeared to be very active in catalyzing the further metabolism of B(u)P phenols. Significant increases in fluorescence were obtained only with low concentrations of peroxide; i.e., concentrations which might only support limited primary oxidation of

REACTIONS

191

B(u)P. As shown in Fig. 3, the optimal cumene hydroperoxide concentration for phenol production was approximately 25 ~.LM and concentrations lower than 25 PM resulted in a decreased activity. The addition of cumene hydroperoxide at a final concentration of 50 ~.LM produced only a small increase in fluorescence intensity during the first minute of incubation. This was followed by an increase in the rate of formation of fluorescent products after 2 min suggesting an increased stability of phenols after significant peroxide has been consumed. Higher concentrations of peroxide (100 PM) resulted in a complete absence of fluorescent products. Under these conditions, almost no phenol was found upon hplc analysis of the ethyl acetate extracts. This biphasic effect of organic peroxide concentration was also noted by Rahimtula and O’Brien (17). When liver microsomes from 5,6-benzoflavone-treated rats were utilized, higher concentrations of cumene hydroperoxide (75 PM) were required for maximal rates of formation of fluorescent products suggesting that these liver microsomes do not support metabolism of the phenols as well as do microsomes from livers of PB-induced animals. The relation between fluorescence of B(u)P products formed during the cumene hydroperoxide-supported hydroxylation reaction and the time of incubation appears to be the result of two processes: the rate of phenol formation (primary oxidation) and the rate BF-induced

FIG. 3. The cumene hydroperoxide-supported, time-dependent formation of benzo(a)pyrene phenols catalyzed by induced rat liver microsomes. One-milliliter aliquots of a standard reaction mixture containing B(a)P, liver microsomes from either PB- or BF-treated rata, and cumene hydroperoxide (at the concentrations indicated) were analyzed for fluorescent phenol products (see Materials and Methods). Zero time points were obtained by adding the hydroperoxide to the chilled reaction mixtures containing 33% acetone.

192

CAPDEVILA,

ESTABROOK,

of phenol degradation. The balance between these two processes is dependent upon the cumene hydroperoxide concentration and the animal pretreatment regimen used prior to isolation of liver microsomal protein. The further oxidation of B(a)P phenols during the steady state of the cumene hydroperoxide-dependent reaction was studied to establish the existence of oneelectron oxidation products of the phenols. As seen in Fig. 4, a large amount of 0x0 radical of 6-hydroxy-B( a)P was noted when one monitored the benzene extract of a reaction mixture containing the 6-phenol, cumene hydroperoxide, and intact microsomes. The ESR spectrum obtained was identical to that generated by the chemical oxidation of the 6-phenol with potassium ferricyanide (3, 4). However, if boiled microsomes were used, there was only 1/60th as much radical formed (Fig. 4A). Figure 4B clearly shows that the small amount of radical formed in the presence of boiled microsomes was identical in shape

AND PROUGH

TABLE III THE EFFECT OF CUMENE HYDROPEROXIDE ON EPOXIDE HYDRASE ACTIVITY

Specific activity0 (nmoI/min/mg protein) Cumene hydroperoxide Substrate

Control

B(a)P-‘l&oxide B(a)P-11,12-oxide Phenanthrene-9,10-oxide

7.3 1.2 121.0

120 /AM 240 /AM 6.9 1.0 119.0

6.3 0.9 112.0

a The incubation mixtures contained liver microsomes from PB-treated rats using the following concentrations of protein B(a)P-7,8-oxide (100 pg protein/80 PI), B(a)P-11,12-oxide (100 pgK?Od), and phenanthrene-9,10-oxide (10 pg/80 ~1).

to that formed by either chemical oxidation of the 6-phenol or the microsomal, organic hydroperoxide-dependent reaction. When either B(a)P or its 3-phenol was used as the substrate, only marginally detectable BG amounts of radical were formed in the presence of cumene hydroperoxide suggesting that the steady-state concentration of oxo radicals (and presumedly the phenols) was very low. Other unstable free radicals may exist during the reaction, but they were not observed under the conditions of our experiments. The activity of epoxide hydrase in liver microsomal fractions from PB-treated rats FIG. 4. ESR spectra of the free radicals formed from was measured using benzo( a)pyrene-‘7,& 6hydroxybenzo(a)pyrene. Reaction mixtures contain- oxide, benzo(a)pyrene-1 1,12-oxide, and phening either native or heat-denatured liver microsomes anthrene-9,10-oxide as substrates (Table from PB-treated animals (0.5 mg/ml), 6-hydroxyB(a)P (100 PM), and cumene hydroperoxide (50 PM) III); the activities were 7.3, 1.2, and 121 formed/min/mg microwere incubated for 2 min at 37°C. The reaction was nmol dihydrodiol terminated by addition of chilled benzene and the somal protein, respectively. In the presence of 120 and 240 PM cumene hydroperoxide, benzene phase treated as described under Materials and Methods section. (A) The intense ESR spectra no dihydrodiol was formed in the absence obtained with native microsomes and the very weak of microsomes or in the presence of tetrasignal formed in the presence of boiled liver micro- hydrofuran-terminated reaction mixtures. somal protein which barely appears above background Cumene hydroperoxide (120 pM) inhibited under these instrumental conditions. The spectra were the hydration of B(a)P ‘I,&oxide, B(a)P normalized for the dilution factors using computer 11,IZoxide, and phenanthrene 9, lo-oxide manipulation. (B) The superimposed ESR spectra of by only 6, 17, and l%, respectively. These the free radicals formed by cumene hydroperoxide in the presence of either native or boiled microsomal results would suggest that at low concentrations (~0.5 mM), cumene hydroperprotein. The ESR spectrum for the boiled protein oxide has little effect on the stability and/or sample in A was amplified 60-fold for comparison in B.

NADPH- AND CLJMENE HYDROPEROXIDE-SUPPORTED

REACTIONS

193

efficiencies and product distribution when one compares liver microsomes from either PB- or BF-treated rats. The differences in catalytic efficiencies with either cytochrome P-450 or P-448 have also been noted by others in the case of acetanilide (20) and benzo( a)pyrene (27). The product ratios obtained with the two systems have been shown to vary for other substrates, such as lauric acid, androstenedione, progesterone, testosterone, and 17/Sestradiol(18, 29). In addition, the ratio of products was highly dependent on the oxidant used as seen in studies using either cumene hydroperoxide, NaIO,, NaC102, pregnenolone 17a-hydroperoxide, t-butyl hydroperoxide, linoleic acid hydroperoxide, and iodosobenzene or iodosobenzene diacetate (18, 29). The existence of products derived from an epoxidation mechanism during the cytochrome P-450- and cumene hydroperoxidedependent aromatic hydroxylation has been demonstrated based on the formation of dihydrodiols and arene oxides of phenanDISCUSSION threne (20) and of the dihydrodiols of benzo(a)pyrene as shown here. However, these This report shows that the NADPH- and products were formed in very low concumene hydroperoxide-dependent metabocentration in the presence of organic perlism of B(a)P have different catalytic oxides relative to the amounts of dihydrodiols produced when NADPH serves as TABLE IV the electron donor. The low amounts of these metabolites formed must be due to CYTOCHROME P-450 DESTRIJC~ION BY CUMENE HYDROPEROXIDE" decreased rates of product formation since we have clearly documented the stability Percentage cytochrome of the dihydrodiols, arene oxides, and P-450 remaining epoxide hydrase in the presence of cumene Reaction time hydroperoxide. Similar epoxidation prod(min) ControP BbP ucts are formed from naphthalene in the chemical hydroxylation systems using Fez+ 0 100 100 salts and organic reducing agents (30) and 1 65 68 from cyclohexene, cyclohexadiene, and cis2 37 59 stilbene using iron-porphine complexes and 3 30 59 iodosylbenzene (31). However, the relaa The reaction was run with reaction mixtures con- tively high amounts of products derived taining 0.5 mg/ml microsomal protein from livers of by oxidation of positions which have high PB-treated rats, 100 ELMcumene hydroperoxide, and electron densities (1, 3, 6, 12) during the either no or 80 PM B(a)P. The reactions were termicumene hydroperoxide-dependent reactions nated by the addition of sodium dithionite and the suggest that the process may be carried out amount of cytochrome P-450 determined using the by a mechanism different than epoxidation. method of Omura and Sato (25). This process most likely involves one* When NADPH was used in place of the organic electron oxidation of benzo( u)pyrene and its peroxide, no destruction of microsomal cytoehrome P-450 was noted during the first 5 min of the reac- phenols to form 0x0 radicals or other radical tion in either the absence or presence of benzo(a)pyrene. species. A similar conclusion has been made activity of epoxide hydrase during a 2-min incubation in the presence of its substrates. In addition, these results indicate that the arene oxide substrates must be reasonably stable in the presence of the organic peroxide since it did not have any significant effect on the rates of hydration of the three arene oxides. As noted in other reports (17,19), cumene hydroperoxide caused a rapid destruction of cytochrome P-450 as revealed by the loss of the Fez+-CO complex of the microsomal cytochrome. Under the conditions described in Table IV, the destruction of the hemoprotein in the presence of 100 PM cumene hydroperoxide was almost complete within the first 2 min of the reaction and addition of B(a)P to the reaction mixture resulted in a significant protective effect. These results suggest that the reaction involving organic peroxides is suicidal in nature compared to the NADPHdependent reaction,

194

CAPDEVILA,

ESTABROOK, AND PROUGH

for the cumene hydroperoxide-dependent oxidation of aminopyrine (32) where stable free radical intermediates have been directly observed. Therefore, the use of organic peroxides to study cytochrome P-450 function for certain substrates may prejudice the conclusions one would make concerning the similarity or differences in the chemical mechanisms operative in these two reactions. These results suggest that during cumene hydroperoxide-dependent hydroxylation of benzo(a)pyrene at least a major portion of the quinones formed, if not all, appears to involve the B( a)P phenols as intermediates. Nagata et al. (3) and Lesko et al. (4) have suggested that the one-electron oxidation of benzo(u)pyrene by microsomes and NADPH leads to the formation of 0x0 radicals and of quinones. It seems likely that during the NADPH-dependent reaction and parallel to reactions leading to epoxidation, active species of oxygen (superoxide anion, hydroperoxides) are formed which may support one-electron oxidation reactions. Since the same quinone products are formed during the cumene hydroperoxide-dependent reaction, one-electron oxidation reactions may be responsible for at least a portion of the quinones produced in the presence of NADPH. Rahimtula and O’Brien (21) have demonstrated that ethanol, npropanol, n-butanol, and n-pentanol are oxidized to their respective aldehydes in the presence of cumene hydroperoxide and either catalase or microsomal cytochrome P-450. Similar products (ketones) are formed during the iron-hydrogen peroxide-dependent oxidation of alkanes and aliphatic alcohols. For example, oxidation by Fez+H,O, lead to a ratio of cyclohexanol to cyclohexanone formed from cyclohexane of 7.0 (33) and a ratio of cyclohexanone to cyclohexanediols formed from cyclohexanol of 4.4 (34). The formation of ketones is thought to be due to metal-catalyzed, free radical autoxidation (one electron) processes. We have shown pronounced differences in the cytochrome P-450-dependent metabolism of benzo( u)pyrene supported by either NADPH or cumene hydroperoxide. The large changes in the position specificity of oxidation by the organic peroxide are

maintained regardless of the animal pretreatment regimen. Since the microsomal protein obtained from BF-treated animals is significantly less active in catalyzing the cumene hydroperoxide-supported reaction, the contribution of the different forms of cytochrome P-450 which exist in liver microsomes will have to be determined using reconstitution experiments with highly purified fractions of the various cytochrome P-450 species. In addition, these results raise a serious question about the “commonness” of the NADPH-supported reactions of cytochrome P-450 and the hydroperoxide-dependent reaction of heme and hemeproteins in general. Since both epoxidation and oneelectron oxidation reactions may coexist during the NADPH-dependent metabolism, the relative contribution of these two reaction mechanisms (which depend both on the chemistry of the substrate and the existence of a multiplicity of hemoproteins) may have significant implications in the carcinogenic and/or toxic properties of various aromatic hydrocarbons. ACKNOWLEDGMENTS The authors are indebted to Wayne Levin, Hoffman-LaRoche Inc. for his assistance in determining epoxide hydrase activity and to Julian Peterson for his helpful criticism and ESR expertise. REFERENCES 1. HOLDER, G., YAGI, H., DANSE~E, P., JERINA, D. M., LEVIN, W., Lu, A. Y. H., AND CONNEY, A. H. (1974) Proc. Nat. Acad. Sci. USA 71, 4356-4360. 2. TOMASZEWSKI, J. E., JERINA, D. M., AND DALY, J. W. (1975) Biochemistry 14, 20242031. 3. NAGATA, C., TAGASHIRA, Y., AND KODAMA, H. (19’74) in Chemical Carcinogenesis (Ts’o, P. 0. P.,

and Di Paolo, J. A., eds), Pt, A, pp. 87- 111, Dekker, New York. 4. LESKO, S., CASPARY, W., LORENTZEN, R., AND Ts’o, P. 0. P. (1975) Bioc~mistry 14, 39783984. 5. YANG, S. K., ROLLER, P. P., Fu, P. P., HARVEY, R. G., AND GELBOIN, H. V. (1977) Biochem. Biophys. Res. Commun. 77, 1176-1182. 6. PROUGH, R. A., PATRIZI, V. W., AND ESTABROOK, R. W. (1976) Cancer Res. 36.4439-4443.

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