0013-7227/90/1266-3101$02.00/0 Endocrinology Copyright © 1990 by The Endocrine Society

Vol. 126, No. 6 Printed in U.S.A.

Estradiol Metabolism by Complementary Deoxyribonucleic Acid-Expressed Human Cytochrome P450s TOSHIFUMI AOYAMA, KENNETH KORZEKWA, KIYOSHI NAGATA, JAMES GILLETTE, HARRY V. GELBOIN, AND FRANK J. GONZALEZ Laboratory of Molecular Carcinogenesis, National Cancer Institute (T.A., H. V.G., F.J.G.), Laboratory of Chemical Pharmacology, National Heart, Lung, and Blood Institute (K.K., K.N., J.G.), National Institutes of Health, Bethesda, Maryland 20892

The roles of P450s IA2, IIA3, and IIIA4 were further established by immunoinhibition using antirat P450 antibodies. Antibody that specifically binds to P450 IIIA3 and IIIA4 inhibited 6070% of estradiol hydroxylation, and antibody against P450 IA2 inhibited 20-40% of the estradiol hydroxylase activity in microsomes from two human liver specimens, suggesting that these enzymes constitute the major forms catalyzing estradiol oxidation in human liver. Immunoinhibition results also suggest that 2-hydroxy- and/or 4-hydroxycatechol estrogens are further metabolized to other yet uncharacterized metabolites by P450s IIIA3 and IIIA4. (Endocrinology 126: 3101-3106, 1990)

ABSTRACT. Twelve forms of human cytochrome P450 were synthesized in human hepatoma Hep G2 cells by means of cDNA-directed expression using vaccinia virus. The cDNAexpressed enzymes were tested for their ability to oxidize estradiol. Incubation of [14C]estradiol with cell lysates containing P450IA2 resulted in the production of 2-hydroxy and 4-hydroxy metabolites with substrate turnovers of 2.74 and 0.27 min"1, respectively. P450s IIIA3 and IIIA4 yielded the same metabolites at about one third the rate of P450 IA2. Low levels of estradiol hydroxylation were also catalyzed by P450s IIC9, IIIA5, and IVBl. Six other P450 forms yielded no detectable metabolism.

/ ^ Y T O C H R O M E P450S1 represent a superfamily of V_y enzymes that carry out monooxidation reactions (1, 2). A small group of P450s participate in pathways of steroidogenesis from cholesterol, including steps leading to the synthesis of mineralocorticoids, glucocorticoids, androgens, and estrogens (3). Other P450s are involved in bile acid synthesis (4) and vitamin D3 hydroxylations (5). A large number of P450s that are expressed in liver and to a lesser extent in extrahepatic tissues are primarily responsible for oxidative degradation of xenobiotics or foreign compounds, such as drugs, environmental pollutants, and toxins. In addition, liver P450s can oxidize endogenous steroids, such as testosterone, estrogen, and their derivatives (6). However, it is generally believed that some P450-mediated metabolism of endogenous steroids are pathways that are not physiologically relevant for steroid degradation and that they occur due to the broad substrate specificities of P450s (2). Indeed, many P450s catalyzing hydroxylations of steroids also metabolize drugs and other chemicals.

Liver P450s are thought to contribute to the inactivation of estrogens, in particular, and to synthetic estrogens used in oral contraceptives (7). Interindividual variation in human estrogen metabolism is believed to play a role in the toxicity or ineffectiveness of the synthetic steroid drug 17a-ethynylestradiol. Moreover, administration of the P450 inducer rifampicin increases estrogen metabolism in women and can result in unwanted pregnancies in those taking oral contraceptives (8). In the current report we used cDNA expression of human P450s to determine which P450 forms are responsible for estrogen metabolism. Twelve forms of human P450 were expressed in cultured human hepatoma cells using vaccinia virus. Six P450s were found to hydroxylate estradiol at its 2 and/or 4 positions; three forms had the highest activity, and these are believed to be the main P450s catalyzing oxidation of this hormone in human liver. Hydroxylation of estrogen by human P450s appears to be a prerequisite for further metabolism of the compound by P450s and perhaps other microsomal enzymes.

Received January 4, 1990. Address requests for reprints to: Dr. Frank J. Gonzalez, Building 37, Room 3E-24, National Institutes of Health, Bethesda, Maryland 20892. 1 The nomenclature used in this report is that described by Nebert

Materials

et al. (1).

Materials and Methods Testosterone and 17a-estradiol were obtained from Sigma Chemical Co. (St. Louis, MO). Hydroxysteroid standards were

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ESTRADIOL OXIDATION BY HUMAN P450

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purchased from Steraloids, Inc. (Wilton, NH). [4-14C]Testosterone (50 mCi/mmol) and [4-14C]estradiol (50 mCi/mmol) were obtained from NEN Research Products (Boston, MA). Vaccinia virus expression of human P450s Recombinant vaccinia viruses containing individual P450 cDNAs were described in earlier reports: P450s IA2 (9), IIA3 (10), IIB7 (11), IIC8 (12), IIC9 (12), IVB1 (13), IIIA4 (14), and IIIA5 (14). Viruses containing the IID6 (15), IIEl (16), IIFl 2 and IIIA3 (see Footnote 2) cDNAs were constructed using protocols developed by Moss and colleagues (17, 18). Hep G2 cells (American Type Culture Collection, Rockville, MD; HB 8065) were used as recipients for recombinant vaccinia virus infection. Wild-type vaccinia strain WR (obtained from B. Moss) was used as a control for non-P450-expressing cells. Confluent plates of Hep G2 cells were infected at a multiplicity of infection of 10 at 0 h, and cells were harvested at 24 h. The cells were washed twice with isotonic NaCl in 0.05 M sodium phosphate buffer, pH 7.4, and then lysed by sonication in a small volume of the same buffer to yield a final protein concentration of about 10 mg/ml. Preparation and immunoinhibition analysis of human liver microsomes Human liver specimens were obtained from the University of Miami, Department of Surgery, Division of Transplantation. Microsomes were prepared as described previously (14). Antibodies were prepared against rat P450d (anti-IA2) and P450PCN1 (anti-IIIAl). The immunoglobulin (IgG) fractions were purified by using protein-A-Sepharose columns, followed by ammonium sulfate precipitation and dialysis into 0.14 M NaCl-0.05 M potassium phosphate buffer, pH 7.4. Immunoinhibition studies were carried out by preincubating IgG with microsomes for 15 min at 40 C before the assay of P450 activities in human liver microsomes. Anti-IIIAl IgG completely inhibited the testosterone 6/3-hydroxylase activities of vaccinia virus-expressed P450s IIIA3, IIIA4, and IIIA5 (see Footnote 2). Anti-IA2 IgG completely inhibited aniline hydroxylase activity catalyzed by vaccinia virus-expressed P450IA2 (see Footnote 2). Enzyme assays For analysis of estradiol metabolism, lysate protein (8 mg) from vaccinia virus-infected Hep G2 cells or human liver microsomes (1.2 mg) was incubated with 1.2 mM NADPH, 100 nM 17/3-estradiol containing [4-14C]estradiol, 50 mM potassium phosphate buffer (pH 7.4), and 1 mM ascorbate in a final a volume of 1 ml. Incubations were carried out at 37 C for 15 min and terminated by the addition of 2 ml dichloromethane. The samples were vigorously vortexed, and the organic phase was removed, filtered, and dried under nitrogen gas. The residue was dissolved in 1:1 methanol-water containing 1.5 mM ascorbate and analyzed immediately. Estradiol metabolites were measured as follows. A Waters 600 solvent delivery system was equipped with a Waters 618 5!

Aoyama, T., H. V. Gelboin, and F. J. Gonzalez, unpublished results.

Endo • 1990 Vol 126-No 6

jum Radial Pak reverse phase column (Waters, Milford, MA) and operated at 2 ml/min at room temperature. The metabolites were separated using a linear gradient between 70:20:10 and 10:80:10 (water-methanol-acetonitrile) over 17 min. The acetonitrile contains 1% acetic acid, giving a constant overall concentration of 0.1%. Detection of metabolites was accomplished with the use of a Waters 490 multiwavelength UV detector, operating at 280 and 242 nm, and a Radiomatic Flowone Beta II radioactivity detector. Quantitation was achieved by electronically integrating and relating the metabolite peak areas and comparing them to the areas of known amounts of pure metabolite standards chromatographed under the same conditions. Aniline p-hydroxylase activities were measured by colorimetric quantitation of p-aminophenol (19). Spectral quantitation of P450 contents were performed using a modification of the procedure of Omura and Sato (20), as described previously (14).

Results Metabolism of estradiol by vaccinia-expressed human P450s Recombinant vaccinia viruses containing individual human P450 cDNAs were constructed and used to infect human hepatoma Hep G2 cells. These cells were chosen as host for P450 expression on the basis of their endogenous expression of sufficient levels of NADPH P450 oxidoreductase to support P450 activity (21, 22). The levels of expression of each P450 were monitored by spectral quantitation, using CO-reduced difference spectra, as described previously (14, 20). P450 contents of vaccinia virus-infected Hep G2 cells ranged from 12-18 pmol/mg total cell protein. Cell lysates from individual recombinant vaccinia virus-infected Hep G2 cells were analyzed for their ability to oxidize estradiol. [14C]Estradiol was incubated with total cell lysates, and the resultant products were resolved by HPLC. HPLC chromatograms of estradiol and its metabolites after incubation with lysates from cells infected with vaccinia viruses containing the IIIA4 cDNA (vIIIA4) and the IA2 cDNA (vIA2) are displayed in Fig. 1. Infected cells expressing P450 IIIA4 and IA2, respectively, revealed three metabolite peaks. Two of these, migrating at 14.5 and 15.5 min on the chromatogram, comigrated with 4-hydroxy- and 2-hydroxyestradiol, respectively. A third metabolite, designated unknown 1 and eluting at 10.5 min, was produced by the IIIA enzymes, but not IA2, and is of unknown structure.3 All metabolites were well separated from estradiol. The results of analysis of 12 recombinant vaccinia viruses containing individual human P450 cDNAs revealed that six forms catalyzed the oxidation of estradiol 3 This metabolite did not comigrate with 6a-hydroxy-, 16a-hydroxy-, or 16/3-hydroxyestradiol.

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ESTRADIOL OXIDATION BY HUMAN P450

A

1 XX

(I)(1)

vfflA4

s

oo ^*

w UJ

a

f It

>—H

> O

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derivative (Fig. 2A). Unknown 1 was found at levels about one third that of 4-hydroxyestradiol. A series of at least three metabolites eluting at later times on the chromatogram (unknowns 2, 3, and 4; Fig. 2A) were also produced. These peaks were not accompanied with UV absorption at 242 and 280 nm, suggesting that they lacked the conjugated A ring. Their structural identities are unknown.

—v^rs

vIA2

B

g

10

15

20

B

25

TIME (min)

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FlG. 1. HPLC chromatogram of estradiol and its metabolites produced by incubation with Hep G2 cells expressing P450 IIIA4 (A) and IA2 (B). The positions of elution of authentic estradiol, 2-hydroxyestradiol (2-OH), and 4-hydroxyestradiol (4-OH) are indicated by vertical arrows. Other peaks are an impurity (I) in the substrate and an unknown metabolite (1). TABLE 1. Assay of estradiol metabolism by vaccinia virus expressed P450s Catalytic activity (nmol/min • nmol P450)

2-OH IA2 IIC9 IIIA3 IIIA4 IIIA5 IVB1

2.74 ± 0.36 ± 0.84 ± 1.30 ± 0.36 ± 0.32 ±

0.36 0.06 0.09 0.20 0.06 0.03

4-OH

Unknown (1)

0.27 ± 004

Estradiol metabolism by complementary deoxyribonucleic acid-expressed human cytochrome P450s.

Twelve forms of human cytochrome P450 were synthesized in human hepatoma Hep G2 cells by means of cDNA-directed expression using vaccinia virus. The c...
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