ARCHIVES

Vol.

OF BIOCHEMISTRY

285, No. 2, March,

Cytochrome Deodutta

Roy,*

*Department Biochemistry

Received

August

AND

BIOPHYSICS

pp. 331-338,

1991

&-Mediated Henry

W. Strobel,t

Redox Cycling of Estrogen’ and Joachim

G. Liehr*,2

of Pharmacology and Toxicology, University of Texas Medical Branch, Galveston, Texas 77550; and TDepartment and Molecular Biology, University of Texas Medical School, Houston, Texas 77225

13, 1990, and in revised

form

November

of

9, 1990

Mammalian hepatic microsomes contain both NADPH-dependent cytochrome P45O/cytochrome P450 reductase and NADH-dependent cytochrome bg/cytochrome b5reductase electron transfer pathways. The role of the cytochrome P450 system in oxidation-reduction reactions for a variety of phenolic and nonphenolic compounds including steroids is well characterized [reviewed by White and Coon (l)], whereas the role of the cyto-

chrome b5 in oxidation-reduction reactions in the course of metabolism of xenobiotics is not as clearly understood. Recently, cytochrome b5 has been shown to exert a stimulatory effect on the NADPH-supported cytochrome P450 monoxygenase-dependent hydroxylation of substrates (24). This stimulatory effect has been attributed to an effector role of cytochrome b5 on one or more than one of the reaction steps in the cytochrome P450 catalytic cycle and/or to an adjunct electron transfer role between the cytochromes. Thus, a role of cytochrome b5 in oxidation or reduction reactions of xenobiotics remains to be defined. In order to develop a more complete picture of the enzymatic contributions to redox reactions of estrogens and estrogen metabolites, we have investigated a role for cytochrome b5 and cytochrome b5 reductase in oxidations and reductions. Microsomal cytochrome P45O/cytochrome P450 reductase-catalyzed redox cycling of diethylstilbestrol (DES)3 and catechol estrogens, the major metabolites of steroidal estrogens, has been demonstrated previously and postulated to play a role in estrogen-induced cancers (5). In this cycle, DES is oxidized to DES Q by the peroxidase activity of microsomal cytochrome P450, whereas DES Q is reduced to stilbene by NADPHdependent cytochrome P450 reductase. The redox cycling has been demonstrated by the accumulation of &Z-DIES as marker product of oxidation and of Z-DES as marker product of reduction (5); for structures see Fig. 7. Redox cycling of estrogens has been studied because of its biological effects (reviewed by Liehr and Roy (6)). This process generates superoxide and semiquinone free radicals (7), which may cause lipid peroxidation and DNA or protein damage and ultimately result in tumor formation and/or cell necrosis (8). These possible biological consequencesof redox cycling of estrogen have prompted this investigation of the major enzyme systems participating

i This work was supported by Grant CA43233 from the National cer Institute, National Institutes of Health (to J.G.L.), and AU1067 from the Welch Foundation (to H.W.S.1. * To whom correspondence should be addressed.

s Abbreviations used: DES, diethylstilbestrol; E-DES or Z-DES, Eor Z-forms (previously trans- or c&forms) of diethylstilbestrol, respectively; DES Q, diethylstilbestrol-4’,4”-quinone; &Z-DIES, Z,Z-dienestrol; (YNF, ol-naphthoflavone or 7,8-benzoflavone.

Previously, we have demonstrated microsomal cytochrome P450-catalyzed redox cycling of estrogens. In this study, we investigated the role of cytochrome bs in redox cycling in order to obtain a full understanding of enzymatic contributions to redox reactions of estrogens. Pure cytochrome P45Oc and hydrogen peroxide or cumene hydroperoxide oxidized diethylstilbestrol (DES) to diethylstilbestrol-4’,4”-quinone (DES Q). This oxidation by H202 was doubled by addition of cytochrome be to cytochrome P45Oc (molar ratio of 1:4), but did not proceed with cytochrome b5 alone. The stimulation by cytochrome ba of the cytochrome P450c-catalyzed oxidation of DES to DES Q occurred via modulation of the V,,,,, of cytochrome P45Oc rather than of the K,,, . DES Q was reduced to DES by purified cytochrome b6 and NADH-dependent cytochrome be reductase. Pretreatment of microsomes with an antibody to cytochrome b6 reductase inhibited microsomal NADH-dependent reduction of DES Q to DES by 55%. Cytochrome be likely participates in the oxidation of DES to DES Q by interacting with cytochrome P45Oc and in the reduction of DES Q to DES by interacting with cytochrome b5 reductase. Thus, the study demonstrates that cytochrome b6 plays an active role in biological oxidation and reduction reactions. 0 1991 Academic Press, Inc.

0003.9861/91 Copyright All rights

$3.00 0 1991 by Academic Press, of reproduction in any form

CanGrant

331 Inc. reserved.

332

ROY, STROBEL,

276

b

8



8



1 450

Wavelength (nm)

-0.003

1 276



3



t

a



’ 450

Wavelength (nm)

AND

LIEHR

aration used was electrophoretically homogenous and had a specific activity of 13.5 nmol/mgprotein. Cytochrome P450 reductase was purified from phenobarbital-induced rat liver microsomes according to the procedure described by Dignam and Strobe1 (13) and had a specific activity of 59.8 bmol of cytochrome c reduced/min/mg protein. Cytochrome bS was purified by the method of Omura and Sato (14) with minor modifications. The specific activity was 47.7 nmol/mg protein. Cytochrome b5 reductase was purified by the method of Schafer and Hultquist (15) and had a specific activity of 224 rmol/min/mg protein. Antibodies to rat cytochrome b5, cytochrome b5 reductase, and cytochrome P450 reductase were raised and purified by the method previously described (11, 16).

FIG. 1. Oxidation

of E-DES to DES Q catalyzed by cytochrome P45Oc (A) and cytochrome P45Oc plus cytochrome b5 (B) in the presence of hydrogen peroxide. The oxidation was monitored by uv spectroscopy. The lowest absorbances were recorded at time 0. An increase in absorbance was recorded every 30 s. There was no increase in absorption in the absence of hydrogen peroxide or enzyme (data not shown).

in the metabolism of estrogens. The present study demonstrates an active role of cytochrome b5 in biological oxidation/reduction reactions of estrogens. MATERIALS

AND

METHODS

Chemicals E-DES, NADPH, ferricytochrome, a-NF, o-naphthoflavone (5,6benzoflavone), bovine serum albulmin, and rabbit IgG were purchased from Sigma Chemical Co. (St. Louis, MO). Z-DES was a gift of Dr. P. Murphy, Eli Lilly and Co. (Indianapolis, IN). DES Q and &Z-DIES were prepared as previously described (9). The purity of DES Q was determined by ultraviolet and nuclear magnetic resonance spectroscopy, high-pressure liquid chromatography and, after rearrangement to Z,ZDIES, by gas chromatography-mass spectrometry. The nature and sources of chemicals used for purifications of enzymes have been discussed in detail previously (10, 11). All solvents and common chemicals used were either analytical grade or of the highest grade available.

Oxidation of DES to Quinone Oxidation of DES to DES Q was catalyzed by (a) cytochrome (b) cytochrome P45Oc plus cytochrome b5, or (c) microsomes.

(a) Cytochrome P45Oc. Incubation conditions of the in vitro oxidation of DES to DES Q by purified cytochrome P45Oc were as follows: The reaction mixture consisted of 10 mM phosphate buffer, pH 7.5, 10 pg/ml dilauroylphosphatidylcholine, O-30 pmol cytochrome P45Oc, DES (O-100 PM), and various concentrations of hydrogen peroxide or cumene hydroperoxide (O-2 mM) in a final volume of 1.0 ml. DES Q formation was monitored by changes in uv spectra for 3 min at room temperature. After 3 min, the reactions were stopped by addition of ice-cold ether, and products were extracted and analyzed by high-pressure liquid chromatography. (b) Cytochrome P45Ocplus cytochrome bs. The incubation conditions were essentially the same as described for cytochrome P45Oc except that various concentrations of cytochrome b5 (2-10 pmol) were added immediately after addition of cytochrome P45Oc. (c) Microsomes. Microsomal catalysis of the oxidation of DES to DES Q was the same as described previously (5) except for the following modifications: The reaction mixture consisted of 1 mg/ml microsomal proteins (microsomes from &naphthoflavone-induced rat liver), 100 pM

TABLE

Oxidation

I

of DES to DES Q by Pure Cytochrome

Instrumentation Ultraviolet spectra were recorded on a Hewlett-Packard Model 8452A diode array spectrophotometer. High-pressure liquid chromatography analyses were carried out using a Waters Associates (Milford, MA) instrument consisting of two solvent delivery systems, Model 510 and Model 501, an automated gradient controller, and a Model 490 multiwavelength detector. Data were recorded by a Waters Model 740 data module.

Animals and Subcellular Fractionations Male Sprague-Dawley rats weighing loo-150 g were used in all experiments. Rats were pretreated with phenobarbital or fl-naphthoflavone as described previously (12). The microsomes from rat livers were prepared by differential centrifugation using the method of Dignam and Strobe1 (13).

Purifications of Enzymes Cytochrome P45Oc” was purified from liver microsomes of /3-naphthoflavone-pretreated rats as described previously (10, 11). The prep-

’ Cytochrome P45Oc is defined as cytochrome P4501Al in accordance with the recommended nomenclature for cytochrome P450 (26).

P45Oc,

Conditions E-DES Z-DES E-DES + H,O, Z-DES + Hz02 Cytochrome P45Oc Cytochrome P45Oc Cytochrome P45Oc Cytochrome P45Oc Cytochrome P45Oc Cytochrome P45Oc Cytochrome P45Oc

+ + + + + + +

HzOz E-DES Z-DES E-DES + H202 Z-DES + Hz02 olNF + E-DES + H20Z aNF + Z-DES + HzO,

P45Oc

Z,Z-DIES (% of stilbenes extracted)

30 28 13 9

< 0.5 < 0.5 < 0.5 < 0.5 < 0.5 < 0.5 < 0.5 (28-32) (25-29) (10-14) (7-10)

Note. Reaction mixtures contained 10 mM phosphate buffer, pH 7.5, dilaurolphosphatidylcholine (10 pg/ml), 20 pmol cytochrome P45Oc, 30 pM E-DES, and 0.8 mM hydrogen peroxide (H202) in the presence or absence of 100 pM aNF in a final volume of 1.0 ml. Reactions were carried out for 3 min at room temperature. Z,Z-DIES, the rearrangement product of DES Q, was extracted and analyzed by high-pressure liquid chromatography as described previously (5). Amounts of product are expressed as percentage of total stilbenes extracted. Values are the means of three experiments; the ranges are given in parentheses.

CYTOCHROME

&-MEDIATED

REDOX

CYCLING

OF

333

ESTROGEN

FIG. 2. Influence of various concentrations of cytochrome P45Oc (left) or cytochrome P45Oc plus cytochrome b5 (right) on the rate of oxidation of E-DES to DES Q. In the latter experiment, data were collected with 20 pmol cytochrome P45Oc and various concentrations of cytochrome bS (1-8 pmol). A control reaction (0.0 value) was carried out without enzymes. The formation of DES Q was monitored by uv spectrophotometry and high-pressure liquid chromatography. Values represent the means of three to four experiments.

DES, 0.8 mM hydrogen peroxide instead of cumene hydroperoxide in a final volume of 1.0 ml of 10 mM phosphate buffer, pH 7.5. Incubations were carried out at room temperature for 10 min. For the antibody-mediated inhibition of oxidation of DES to DES Q, microsomes (1 mg/ml) were incubated with the antibody to cytochrome b5 (4 mg/ml) for 60 min at 37°C. Subsequently, microsomes were isolated by differential centrifugation as described above and washed with 10 mM phosphate buffer, pH 7.5, containing 1 mM EDTA. Antibody-mediated inhibition of oxidation of DES to DES Q was carried out using these antibody-containing microsomes as described. Control reactions were carried out in the presence of bovine serum albumin or IgG

(4 mdml). Ultraviolet Monitoring of the Oxidation of DES to DES Q The conversion of DES to DES Q was monitored as a gradual increase in uv absorption in the range 275-450 nm recorded every 30 s for 3 min. The identity of DES Q was confirmed by matching with uv absorption characteristics of synthetic DES Q. The rate of formation of DES Q was calculated by using synthetic DES Q as a standard.

-504

0

25 :

HPLC Analyses of the Rearrangement Product of DES Q Z,Z-DIES, Q formation chromatography

the rearrangement product of DES Q and marker (5), was extracted and analyzed by high-pressure as described previously (5).

Reduction of DES Q to Z-DES The reduction of DES Q to Z-DES, a marker product of reduction (6) was catalyzed by (a) cytochrome P450 reductase, (b) cytochrome b5/ cytochrome b5 reductase, and (c) microsomes and was analyzed by gradual disappearance of ultraviolet absorptions of DES Q as monitored by spectrophotometry. (a) Cytochrome P450 reductase system. The reaction conditions for the reduction of DES Q as described by Roy and Liehr (7) were used. (b) Cytochrome b&ytochrome bS reductase. The reaction conditions were the same as used with cytochrome P450 reductase, except that cytochrome b5 and cytochrome b5 reductase were used as reducing enzymes, and 0.2 mM NADH was used as a cofactor.

02;>>

-0.09

of DES liquid

,

-0.05

0.00

0.05

0.09

l/DES(l/S) FIG. 3. Influence of various substrate concentrations on the rate of oxidation of E-DES to DES Q. The data are presented as a double-reciprocal plot and as substrate-dependent product formation (inset). Incubations were carried out in the presence of cytochrome b6 (5 pmol) (A), cytochrome P45Oc (20 pmol) (O), or cytochrome P45Oc plus cytochrome b6 (20 pmol and 5 pmol, respectively) molar ratio (0). Values represent the means of three to four experiments.

334

ROY,

STROBEL,

AND

fc) Microson& reduction. The microsomal system consisted of 1 mg/ ml microsomal protein, 0.2 mM NADPH or NADH, 50 pM DES Q in a final volume of 1.0 ml of 10 mM phosphate buffer, pH 7.5. For the antibody-mediated inhibition of reduction of DES Q to DES, microsomes (1 mg/ml) were incubated with the antibody to cytochrome P450 reductase or cytochrome b, reductase (4 mg/ml) for 60 min at 37’C. Subsequently, microsomes were isolated as described above and reactions were carried out as described previously (7). Control reactions were carried out in the presence of bovine serum albumin or IgG (4 w/ml).

Product Analysis The reduction of DES Q was monitored spectrophotometrically by measuring the decrease in absorption at 312 nm (7). Products were extracted and analyzed by high-pressure liquid chromatography as described previously (7). The formation of reduction product Z-DES was expressed as a percentage of the total stilbenes extracted.

RESULTS

Oxidation of DES to DES Q The oxidation of DES to DES Q is mediated by hydrogen peroxide and cytochrome P45Oc purified from rat liver (Fig. 1A and Table I). Thus, this isoform of cytochrome P450 is identified as one of the enzymes previously characterized as microsomal peroxidatic activity of cytochrome P450. aNF, a known specific inhibitor of cytochrome P45Oc (ll), inhibited this oxidation by 65% (Table I). The oxidation to DES Q catalyzed by purified enzyme is dependent on reaction time (Fig. 1A) and concentrations of enzyme (Fig. 2, left panel), substrate (Fig. 3), and the cofactors hydrogen peroxide or cumene hydroperoxide (Fig. 4). The rate of oxidation of DES to DES Q more than doubled when catalyzed by a mixture of pure cytochrome b5 (20%) and cytochrome P45Oc (80%) (Fig. 1B and Fig. 3). Under the reaction conditions described above, the

-40

TABLE Stimulatory

II

Effect of Cytochrome b5 on the Oxidation to DES Q by Pure Cytochrome P45Oc

of DES

Z,Z-DIES (% of stilbenes extracted)

Conditions Cytochrome b5 + E-DES Cytochrome b5 + Z-DES Cytochromes b5 + P45Oc Cytochromes b6 + P45Oc Cytochrome b5 + E-DES Cytochrome b6 + Z-DES Cytochromes b5 + P45Oc Cytochromes b5 + P45Oc Bovine serum albumin + + E-DES + H202 Bovine serum albumin + + Z-DES + HrOr

+ H202 + Hz02 + Hz02 + E-DES + HrO, + Z-DES + H1Or cytochrome P45Oc

< 0.5 < 0.5 < 0.5 < 0.5 < 0.5 < 0.5 48 (44-52) 45 (41-46) 32 (30-36)

cytochrome

P45Oc 30 (28-32)

Note. Reaction conditions were the same as described in Table I, except that 5 pmol cytochrome bS was added. Amounts of oxidation product, Z,Z-DIES, are expressed as percentage of total stilbenes extracted. Values are the means of three experiments; ranges are given in parentheses.

kinetic parameters are V,,,,, = 0.37 nmol for the reaction catalyzed by cytochrome P45Oc and V,,, = 0.87 nmol for the reaction catalyzed by cytochrome P45Oc plus cytochrome b5 (4:l) with a K, = 13 and 18 PM, respectively (Fig. 3 and Table VII). An increase in the molar ratio of cytochromes bJP450c beyond 0.2-0.3 increased the reaction rate only marginally (Fig. 2, right panel). The increase in the rate of oxidation of DES to DES Q, when catalyzed by a mixture of pure cytochromes b5 and P45Oc compared to cytochrome P45Oc alone was con-

-20

20

1 /PercmLk FIG. 4. Influence of various concentrations Q. The data are presented as a double-reciprocal to four experiments.

LIEHR

40

(mM >

of hydrogen peroxide (0) or cumene hydroperoxide plot and as cofactor-dependent product formation

(0) on the rate of oxidation (inset). Values represent

of E-DES the means

to DES of three

CYTOCHROME TABLE Microsomal

Oxidation

REDOX

III of DES

Conditions Microsomes Microsomes Microsomes Microsomes Microsomes Microsomes Microsomes Microsomes Microsomes Cytochrome Cytochrome Microsomes + E-DES Microsomes Microsomes + E-DES

&-MEDIATED

+ E-DES + Z-DES + H202 + E-DES + Hz02 + (uNF + E-DES + H202 + Z-DES + H202 + cvNF + Z-DES + HzOz + cytochrome b5 antibody b, antibody b5 antibody + E-DES + cytochrome b6 antibody + HzOz + IgG + E-DES + HzOz + bovine serum albumin + H,OB

to DES

Q

Oxidation to Z,Z-DIES (76 of stilbene extracted)

65 25 63 19

< 0.5 < 0.5 < 0.5 < 0.5 (60-68) (20-26) (58-65) (15-20) < 0.5 < 0.5 < 0.5

49 (45-50) 67 (60-69) 67 (60-68)

Note. Reaction conditions were the same as described previously (5), except that P-naphthoflavone-induced rat liver microsomes (1 mg/ml), 100 pM E-DES, and 0.8 mM Hz02 cofactor were used. Reactions were carried out for 10 min at room temperature. The oxidation product, Z,Z-DIES, was extracted and analyzed by HPLC as described previously (5). Amounts of oxidation product are expressed as percentage of total stilbenes extracted. Values are the means of four to six experiments; the ranges are given in parentheses.

firmed by analysis of the formation of marker product of oxidation, Z,Z-DIES (Table II). DES Q is unstable in protic solvents and spontaneously rearranges to Z&DIES (9). After incubation of DES (30 PM) with 20 pmol pure cytochrome P45Oc and hydrogen peroxide, 200 pmol Z,ZDIES/min was isolated. On the other hand, when 5 pmol cytochrome b5 was added to the incubation mixture, the yield of &Z-DIES increased by 60% (Table II). E- or Z-DES was oxidized to DES Q by rat liver microsomes and hydrogen peroxide in a facile manner (Table

Wavelength (nm)

CYCLING

OF

III) as shown previously (5). The participation of cytochrome b5 in these microsomal oxidations was demonstrated by oxidations with microsomes preincubated with antibody to cytochrome b6. In these reactions, DES Q formation was inhibited by 25% (Table III). This inhibition was not observed in control incubations with bovine serum albumin or IgG. Although both organic hydroperoxide and hydrogen peroxide may serve as cofactor for the cytochrome P45Ocmediated oxidation of DES in the presence or absence of cytochrome b5, DES Q formation is more efficient in the presence of cumene hydroperoxide (K, = 0.14 mM) than hydrogen peroxide (K, = 1.1 mM) (Fig. 4). Optimal DES Q formation is achieved with approximately 0.5 mM cumene hydroperoxide. The product yield decreases with increasing organic hydroperoxide cofactor concentrations presumably due to destruction of the enzyme. With NADPH as cofactor in incubations containing cytochrome P45Oc and cytochrome P450 reductase, oxidation to DES Q could not be detected, presumably, because of facile reduction of DES Q catalyzed by cytochrome P450 reductase. These results are in agreement with results obtained previously (5). In summary, the rate of oxidation of DES to quinone is more than doubled when catalyzed by a mixture of cytochromes b5 and P45Oc in a molar ratio of 1:4. This doubling is not additive since cytochrome b5 alone does not catalyze this metabolic reaction. Rather, the enhanced oxidation of stilbene by this mixture of cytochromes b5 and P45Oc demonstrates the participation of an additional pathway of electron flow during metabolic oxidation. The participation of cytochrome b5 in oxidations is demonstrated by the inhibition of the conversion of DES to Z,ZDIES in the presence of antibody to cytochrome b5. Reduction

of DES Q

The reduction of DES Q is accomplished by a mixture of 0.165 nmol cytochrome b5 and 204 pmol cytochrome b5 reductase and NADH (Figs. 5 and 6, and Tables V and

Wavelength (nm)

FIG. 5. Reduction of DES Q to Z- and E-DES catalyzed by cytochrome Control incubations were carried out in the absence of enzyme (A) or cofactor The highest absorbances were recorded at time 0. A decrease in absorbance

335

ESTROGEN

b5 reductase (B) or cytochrome (data not shown). The reductions was recorded every 15 s.

Wavelength (nm) b5 reductase plus cytochrome b5 (C). were monitored by uv spectroscopy.

336

ROY,

STROBEL,

AND

LIEHR

0.8 1

0:8

0:4

0:4 l/DES

0.8

Q (IN)

FIG. 6. Influence of various substrate concentrations on the rate of reduction of DES Q to Z- and E-DES catalyzed reductase (O), cytochrome b5 reductase plus cytochrome b5 (O), or cytochrome b5 reductase (A). The data are presented plot and as substrate-dependent product formation (inset). Values represent the means of three to four experiments.

VII). Kinetic parameters are K, = 8.0 PM and V,,, = 9.4 nmol/min. Both protein components are necessary elements for electron flow and reduction of the quinone since cytochrome b5 reductase or cytochrome b5 alone possess much lower or no reducing activity, respectively (Table V). The cytochrome P450 reductase-catalyzed reduction of DES Q to parent stilbene, described previously (K, = 11.9 PM) (7), was confirmed in this study (& = 11.0 PM) for purposes of validation and comparison (Figs. 5 and 6, and Tables IV and VII). The rate of reduction by cytochrome b5 and cytochrome b5 reductase is approximately one-third of the value obtained with cytochrome

P450 reductase (Tables IV, V, and VII). The reduction of DES Q by cytochrome bSplus cytochrome b5 reductase was confirmed further by analysis of reaction products in the presence of antibody to cytochrome b5reductase (Table VI). Under these reaction conditions, an inhibition by 55% of formation of Z-DES was observed. DISCUSSION

The data reported here demonstrate that cytochrome b5 plays an active role in the redox cycling of estrogens. TABLE

Reduction TABLE

In Vitro Reduction by Cytochrome

IV

of DES Q to Z-DES P450 Reductase

DES Q Cytochrome P450 reductase DES Q + NADPH Cytochrome P450 reductase Cytochrome P450 reductase + NADPH

+ DES

Q

+ DES

Q

< < <

Cytochrome b5-mediated redox cycling of estrogen.

Previously, we have demonstrated microsomal cytochrome P450-catalyzed redox cycling of estrogens. In this study, we investigated the role of cytochrom...
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