Roles of cytochromes P450 1A2 and 3A4 in the oxidation of estradiol and estrone in human liver microsomes

Article abstract of DOI:10.1021/tx970217f

Of seven cDNA-expressed human cytochrome P450 (P450) enzymes (P450s 1A2, 2B6, 2C9, 2C19, 2D6, 2E1, and 3A4) examined, P450 1A2 was the most active in catalyzing 2- and 4-hydroxylations of estradiol and estrone. P450 3A4 and P450 2C9 also catalyzed these reactions although to lesser extents than P450 1A2. P450 1A2 also efficiently oxidized estradiol at the 16α-position but was less active in estrone 16α-hydroxylation; the latter reaction and also estradiol 16α-hydroxylation were catalyzed by P450 3A4 at significant levels. Anti-P450 1A2 antibodies inhibited 2- and 4-hydroxylations of these two estrogens catalyzed by liver microsomes of some of the human samples examined. Estradiol 16α-hydroxylation was inhibited by both anti-P450 1A2 and anti-P450 3A4, while estrone 16α-hydroxylation was significantly suppressed by anti-P450 3A4 in human liver microsomes. Fluvoxamine efficiently inhibited the estrogen hydroxylations in human liver samples that contained high levels of P450 1A2, while ketoconazole affected these activities in human samples in which P450 3A4 levels were high. α- Naphthoflavone either stimulated or had no effect on estradiol hydroxylation catalyzed by liver microsomes; the intensity of this effect depended on the human samples and their P450s. Interestingly, in the presence of anti-P450 3A4 antibodies, α-naphthoflavone was found to be able to inhibit estradiol and estrone 2-hydroxylations catalyzed by human liver microsomes. The results suggest that both P450s 1A2 and 3A4 have major roles in oxidations of estradiol and estrone in human liver and that the contents of these two P450 forms in liver microsomes determine which P450 enzymes are most important in hepatic estrogen hydroxylation by individual humans. P450 3A4 may be expected to play a more important role for some of the estrogen hydroxylation reactions than P450 1A2. Knowledge of roles of individual P450s in these estrogen hydroxylations has relevance to current controversies in hormonal carcinogenesis.

Full text of DOI:10.1021/tx970217f

Chem. Res. Toxicol. 1998, 11, 659-665
659
Roles of Cytoch r om es P 450 1A2 a n d 3A4 in th e Oxid a tion
of Estr a d iol a n d Estr on e in Hu m a n Liver Micr osom es
Hiroshi Yamazaki,§ Peter M. Shaw,† F. Peter Guengerich,‡ and
Tsutomu Shimada*,§
Osaka Prefectural Institute of Public Health, 3-69 Nakamichi 1-chome, Higashinari-ku,
Osaka 537, J apan, PanVera Corporation, Madison, Wisconsin 53711, and Department of
Biochemistry and Center in Molecular Toxicology, Vanderbilt University School of Medicine,
Nashville, Tennessee 37232
Received December 8, 1997
Of seven cDNA-expressed human cytochrome P450 (P450) enzymes (P450s 1A2, 2B6, 2C9,
2C19, 2D6, 2E1, and 3A4) examined, P450 1A2 was the most active in catalyzing 2- and
4-hydroxylations of estradiol and estrone. P450 3A4 and P450 2C9 also catalyzed these
reactions although to lesser extents than P450 1A2. P450 1A2 also efficiently oxidized estradiol
at the 16R-position but was less active in estrone 16R-hydroxylation; the latter reaction and
also estradiol 16R-hydroxylation were catalyzed by P450 3A4 at significant levels. Anti-P450
1A2 antibodies inhibited 2- and 4-hydroxylations of these two estrogens catalyzed by liver
microsomes of some of the human samples examined. Estradiol 16R-hydroxylation was
inhibited by both anti-P450 1A2 and anti-P450 3A4, while estrone 16R-hydroxylation was
significantly suppressed by anti-P450 3A4 in human liver microsomes. Fluvoxamine efficiently
inhibited the estrogen hydroxylations in human liver samples that contained high levels of
P450 1A2, while ketoconazole affected these activities in human samples in which P450 3A4
levels were high. R-Naphthoflavone either stimulated or had no effect on estradiol hydroxy-
lation catalyzed by liver microsomes; the intensity of this effect depended on the human samples
and their P450s. Interestingly, in the presence of anti-P450 3A4 antibodies, R-naphthoflavone
was found to be able to inhibit estradiol and estrone 2-hydroxylations catalyzed by human
liver microsomes. The results suggest that both P450s 1A2 and 3A4 have major roles in
oxidations of estradiol and estrone in human liver and that the contents of these two P450
forms in liver microsomes determine which P450 enzymes are most important in hepatic
estrogen hydroxylation by individual humans. P450 3A4 may be expected to play a more
important role for some of the estrogen hydroxylation reactions than P450 1A2. Knowledge
of roles of individual P450s in these estrogen hydroxylations has relevance to current
controversies in hormonal carcinogenesis [Service, R. F. (1998) Science 279, 1631-1633].
Estradiol and estrone, two major estrogens that are
distributed widely in mammalian tissues, have been
shown to be oxidized at several positions by different
P450 enzymes (9, 10). Catechol estrogens, the products
of estrogens formed by 2- and 4-hydroxylations by P450
enzymes, have been implicated as important in breast
cancer. These metabolites can serve as intermediates for
the generation of more reactive semiquinones and quino-
nes (11-13). Hydroxylation of estradiol by P450 enzymes
at the 16R-position has also been postulated to be
important in mammary carcinogenesis because 16R-
hydroxyestradiol can bind covalently to estrogen recep-
tors (11, 14-16). Exactly which of these pathways is
most important to estrogen-induced cancers remains
controversial.
In tr od u ction
Cytochrome P450 (P450) comprises a superfamily of
enzymes that catalyze oxidation of a great number of
xenobiotic chemicals such as drugs, toxic chemicals, and
carcinogens as well as endobiotic chemicals including
steroids, fatty acids, prostaglandins, and lipid-soluble
vitamins (1-3). Several steroid hormones have been
shown to be catalyzed by P450 enzymes localized in
endoplasmic reticulum (microsomal) membranes of mam-
malian livers (4, 5). Testosterone and progesterone have
been shown to be oxidized mainly by the P450 3A
subfamily of enzymes to 6â-, 2â-, and 15â-hydroxytest-
osterone and to 6â-, 16R-, and 2â-hydroxyprogesterone,
respectively, in human liver microsomes (5-7), and we
have recently shown that hydroxylation of testosterone
at the 17-position (to form androstenedione) and hy-
droxylation of progesterone at the 21-, 16R-, and 17R-
positions are catalyzed mainly by P450 2C19 and to lesser
extents by P450 2C9 (8).
Studies have been done in several laboratories to
identify which P450 enzymes are involved in the oxida-
tion of estrogens in humans (9, 10, 17, 18). P450 enzymes
in mammalian tissues have generally been recognized to
be more active in catalyzing estrone and estradiol at the
C-2 than the C-4 position; in humans, the former reac-
tions are reported to be catalyzed by several P450
enzymes including P450s 1A1, 1A2, and 3A4 (17-19).
* Address correspondence to: Dr. T. Shimada. Fax: (81) 6-972-2393.
E-mail: shimada@iph.pref.osaka.jp.
§ Osaka Prefectural Institute of Public Health.
† PanVera Corp.
‡ Vanderbilt University School of Medicine.
S0893-228x(97)00217-8 CCC: $15.00 © 1998 American Chemical Society
Published on Web 05/16/1998

660 Chem. Res. Toxicol., Vol. 11, No. 6, 1998
Yamazaki et al.
There are also complicated results suggesting that sev-
eral human P450 enzymes can be involved in the 16R-
hydroxylation of estrogens, although definitive studies
to indicate which P450 enzymes are primarily involved
in the oxidations of these estrogens are lacking (17, 18).
Recently estradiol 4-hydroxylation has been reported to
be catalyzed by P450 1B1 (20). Other studies have shown
that estradiol 4-hydroxylation is catalyzed mainly by
P450 3A enzymes in human liver, since anti-P450 3A4
antibodies can inhibit the microsome-catalyzed 4-hy-
droxylation of estradiol by 60-80% (9, 17, 19, 21).
The present study was, therefore, undertaken to
determine which P450 enzymes are most active in the
hydroxylations of estradiol and estrone at different
positions. Initial studies were done using recombinant
human P450 enzymes, and further studies were done in
the context of human liver microsomes in order to put
the work with recombinant enzymes into perspective.
F igu r e 1. Estradiol hydroxylation by cDNA-expressed human
P450 enzymes and by human liver microsomal samples HL-C6,
HL-C15, and HL-C19. The substrate concentration used was
100 µM, and the P450 concentration was 0.2 µM for recombinant
P450 enzymes and 1 µM for liver microsomes. Data are means
of duplicate determinations.
Exp er im en ta l P r oced u r es
Ch em ica ls. Estrone, estradiol, and their hydroxylated
metabolites were purchased from Sigma Chemical Co. (St. Louis,
MO). Other chemicals and reagents used in this study were
obtained from sources described previously or were of highest
quality commercially available (8, 22, 23).
En zym e P r ep a r a tion s. Recombinant P450s 2C9, 2C19,
2D6, and 3A4 expressed in microsomes of Trichoplusia ni cells
infected with a baculovirus containing human P450 and the
human NADPH-P450 reductase cDNA inserts were prepared
as described (24, 25). Other recombinant P450 enzymes includ-
ing P450s 1A2, 2B6, and 2E1 in a baculovirus system coex-
pressing human NADPH-P450 reductase were obtained from
Gentest Co. (Woburn, MA); the P450 contents in these latter
systems were used as described in the data sheets provided by
the manufacturer.
Human liver samples were obtained from organ donors or
patients undergoing liver resection as described previously (26,
27). Liver microsomes were suspended in 10 mM Tris-Cl buffer
(pH 7.4) containing 1.0 mM EDTA and 20% glycerol (v/v) (28).
Human P450s 1A2, 2C9, and 3A4 were purified to electro-
phoretic homogeneity from liver microsomes as described (9, 29,
30). Rabbit anti-human P450 1A2, 2C9, and 3A4 antibodies
were prepared as described (30, 31).
F igu r e 2. Estrone hydroxylation by cDNA-expressed human
P450 enzymes and by liver microsomes of human samples HL-
C6, HL-C15, and HL-C19. Other details are the same as in the
legend to Figure 1.
En zym e Assa ys. Estradiol and estrone hydroxylation ac-
tivities were determined as described (17-19), with slight
modifications. The standard incubation mixture (final volume
of 0.25 mL) contained liver microsomes (1.0 µM P450) or
recombinant P450 enzymes (0.2 µM), 50 mM potassium phos-
phate buffer (pH 7.4), an NADPH-generating system (32), and
estradiol or estrone (100 µM). Incubations were carried out at
37 °C for 30 min and terminated by adding 1.5 mL of CH2Cl2
and 0.3 M NaCl. The organic phase was taken to dryness under
a nitrogen stream at 37 °C. The residue was dissolved in a
mixture of 33% CH3CN (v/v) in 1.0% acetic acid (v/v). Product
formation was determined by HPLC with a C18 (5 µm) analytical
column (4.6 × 150 mm; Kanto Chemical, Tokyo). The elution
was conducted with a mixture of 33% CH3CN (v/v) in 1.0% acetic
acid (v/v) at a flow rate of 1.0 mL/min, and detection was by
UV absorbance at 280 nm. We did not add ascorbic acid in the
HPLC mobile phase nor in the incubation mixture, since no
difference in the hydroxylated metabolites of estrogens was
found during assays in this system (17). Most assays were done
in duplicate and are representative of other confirmatory
studies.
dodecyl sulfate-polyacrylamide gel electrophoresis/immunochem-
ical development (“Western blotting”) (35).
Kinetic parameters for substrate oxidations by recombinant
P450 enzymes and human liver microsomes were estimated
using a computer program (KaleidaGraph program from Syn-
ergy Software, Reading, PA) designed for nonlinear regression
analysis.
Resu lts
Estr a d iol a n d Estr on e Hyd r oxyla tion s by cDNA-
Exp r essed Hu m a n P 450 En zym es. Seven forms of
cDNA-expressed human P450 enzymes including P450
1A2, P450 2B6, P450 2C9, P450 2C19, P450 2D6, P450
2E1, and P450 3A4, prepared in baculovirus systems,
were used to determine which P450 enzymes catalyze
estradiol and estrone hydroxylations significantly (Fig-
ures 1 and 2). Estrogen oxidation activities by human
liver microsomal samples HL-C6, HL-C15, and HL-C19
were also determined. These three microsomal prepara-
tions contained total spectrally determined P450 levels
(nmol/mg of microsomal protein) of 0.66, 0.42, and 0.69,
Oth er Assa ys. Concentrations of P450 and protein were
estimated spectrally by the methods of Omura and Sato (33)
and Lowry et al. (34), respectively. The contents of human P450
proteins in liver microsomes were estimated by coupled sodium

Oxidation of Estradiol and Estrone by Human P450s
Chem. Res. Toxicol., Vol. 11, No. 6, 1998 661
F igu r e 3. Effects of preimmune IgG and anti-P450 antibodies (5 mg of IgG/nmol of microsomal P450) on oxidations of estradiol (A,
B, and C) and estrone (D, E, and F) catalyzed by human liver microsomes (sample HL-C19). Data are means of duplicate
determinations. Control activities (nmol of products formed/min/nmol of microsomal P450) in the absence of antibodies were 0.72,
0.051, and 0.069 for 2-, 4-, and 16R-hydroxylations of estradiol and 0.52, 0.045, and 0.075 for 2-, 4-, and 16R-hydroxylations of estrone,
respectively. Abbreviations used in the figure: 2-OH, 2-hydroxylation; 4-OH, 4-hydroxylation; 16R-OH, 16R-hydroxylation.
Ta ble 1. Con cen tr a tion Dep en d en ce of Estr a d iol a n d
respectively. Microsomal sample HL-C6 had P450 1A2,
P450 2C9, and P450 3A4 levels of 5%, 12%, and 73% total
P450, respectively, as judged by immunoblotting analysis.
Estr on e Hyd r oxyla tion s by Recom bin a n t P 450 1A2 a n d
P 450 3A4 Exp r essed in Ba cu lovir u s System sa
2-hydroxylation
4-hydroxylation 16R-hydroxylation
Sample HL-C15 contained P450 1A2, 2C9, and 3A4 levels
of 23%, 21%, and 14%, respectively, and sample HL-C19
had levels of 25%, 16%, and 46% total P450, respectively.
Of the seven cDNA-expressed human P450 enzymes
examined, the P450 1A2 system was the most active in
catalyzing estradiol 2-, 4-, and 16R-hydroxylations, when
determined at a substrate concentration of 100 µM
(Figure 1). P450s 2C9 and 3A4 catalyzed estradiol 2- and
4-hydroxylations at lower rates than did P450 1A2.
Several P450 enzymes including P450s 1A2, 2C9, 2C19,
2D6, and 3A4 oxidized estradiol at the 16R-position.
Human liver microsomal activities for estradiol hydroxy-
lations were lower than those catalyzed by P450 1A2.
Similar patterns of activities of estradiol hydroxyla-
tions by recombinant human P450 enzymes were ob-
served when estrone was used for the substrate (at 100
µM), except that estrone 16R-hydroxylation activities
were highest with P450 3A4 followed by P450s 2C9 and
2C19 (Figure 2). The catalytic activity of P450 1A2 for
estrone 16R-hydroxylation was very low.
Con cen tr a tion Dep en d en ce of Oxid a tion of Es-
tr a d iol a n d Estr on e by Hu m a n P 450 En zym es.
Steady-state kinetic analysis was done for hydroxylations
of estradiol and estrone by recombinant P450 1A2 and
P450 3A4 systems (Table 1). In all cases, the Km values
were in the range of 10-120 µM, so most of the subse-
quent experiments were done at the single substrate
concentration of 100 µM. With P450 1A2, the rank order
of Vmax for estradiol and estrone hydroxylation was 2 >
4 ∼ 16R. With P450 3A4, the rank order was 2 ∼ 4 ∼
16R for estrone. The substrate concentrations used in
many of the incubations were 100 µM (both estradiol and
estrone); the levels are much higher than those seen in
the physiological state. However, the Km values for the
three hydroxylations are relatively high for P450s 1A2
and 3A4 (Table 1), and even a concentration of 100 µM
is not saturating.
Km
Vmax
Km
Vmax
Km
Vmax
(µM)
(min-1
)
(µM)
(min-1
)
(µM)
(min-1
)
Estradiol Hydroxylation
11 ( 2 28 ( 6
P450 1A2 20 ( 6
0.9 ( 0.1 58 ( 4 0.7 ( 0.2
P450 3A4 54 ( 11 0.8 ( 0.2 111 ( 42 0.3 ( 0.2 75 ( 18 0.4 ( 0.2
Estrone Hydroxylation
9.2 ( 2.1 17 ( 5
P450 1A2 19 ( 6
2.0 ( 0.3
<0.1
P450 3A4 102 ( 30 0.7 ( 0.4 78 ( 26 0.6 ( 0.3 64 ( 8 0.5 ( 0.2
a
Kinetic parameters were determined at substrate concentra-
tions between 10 and 150 µM and P450 concentrations of 0.04 µM
for P450 1A2 and 0.2 µM for P450 3A4. Incubation time used for
P450 1A2 and 3A4 was 15 and 30 min, respectively. Results are
presented as means ( SE.
HL-C19 (Figure 3). Anti-P450 1A2 significantly inhibited
the 2-hydroxylation of estradiol and estrone, 4-hydroxy-
lation of estradiol and estrone, and 16R-hydroxylation of
estradiol. In contrast, estrone 16R-hydroxylation was
markedly suppressed by anti-P450 3A4; the antibody also
inhibited 2-, 4-, and 16R-hydroxylations of estradiol.
Effects of anti-P450 2C9 antibodies were less than those
of anti-P450 1A2 and anti-P450 3A4 in the estrogen
hydroxylations catalyzed by liver microsomes.
Effects of Ch em ica l P 450 Mod ifier s on Hyd r oxy-
la tion s of Estr a d iol a n d Estr on e by Hu m a n Liver
Micr osom es. The results obtained using cDNA-ex-
pressed P450s suggest that several P450 enzymes includ-
ing P450s 1A2, 3A4, and 2C9 are involved in the
hydroxylations of estradiol and estrone by human liver
microsomes. However, these systems may not be directly
comparable, and in addition, the results must be consid-
ered in terms of the expression levels of individual P450s
in liver. Therefore, we utilized several human liver
microsomal samples to further investigate P450 roles.
Ketoconazole (inhibitor of P450 3A4), sulfaphenazole
(inhibitor of P450 2C9), fluvoxamine (inhibitor of P450
1A2), and R-naphthoflavone (inhibitor of P450 1A2 and
sometimes activator of P450 3A4) were examined for
their effects on estrogen hydroxylation in human liver
microsomes.
Effects of An ti-P 450 An tibod ies on Hyd r oxyla -
t ion s of E st r a d iol a n d E st r on e b y Hu m a n Liver
Micr osom es. Antibodies raised against P450 1A2, P450
2C9, and P450 3A4 were used to inhibit estradiol and
estrone hydroxylations catalyzed by human microsomal
When liver microsomal sample HL-C6 (a sample high
in P450 3A4) was used, ketoconazole caused very sig-

662 Chem. Res. Toxicol., Vol. 11, No. 6, 1998
Yamazaki et al.
F igu r e 4. Effects of ketoconazole (O), sulfaphenazole (b), fluvoxamine (0), and R-naphthoflavone (9) on estradiol 2-hydroxylation
(A and B), 4-hydroxylation (C and D), and 16R-hydroxylation (E and F) catalyzed by liver microsomes of human samples HL-C6 (A,
C, and E) and HL-C19 (B, D, and F). Different concentrations of chemicals were added to the incubation mixtures as indicated. Data
are means of duplicate determinations. Control activities (nmol of products formed/min/nmol of microsomal P450) of 2-, 4-, and
16R-hydroxylations of estradiol in the absence of chemicals were 0.53, 0.049, and 0.076 for liver microsomal preparation HL-C6 and
0.56, 0.048, and 0.069 for sample HL-C19, respectively.
estrone 2- and 4-hydroxylations by liver sample HL-C19
microsomes. R-Naphthoflavone did not affect estrone 2-
and 16R-hydroxylations but significantly enhanced es-
trone 4-hydroxylation catalyzed by liver microsomes.
Effects of Com bin a tion s of An ti-P 450 An tibod ies
a n d Ch em ica l P 450 Mod ifier s on Hyd r oxyla tion s of
Estr adiol an d Estr on e by Hu m an Liver Micr osom es.
The effects of chemical P450 modifiers on the estrogen
hydroxylations by human liver microsomes were exam-
ined in the presence or absence of anti-P450 1A2 and
anti-P450 3A4. In the presence of anti-P450 1A2, estra-
diol hydroxylations were completely inhibited by keto-
conazole and were stimulated by R-naphthoflavone in
liver microsomes of a sample containing a high level of
P450 3A4 (HL-C6) (Table 2). In the presence of anti-
F igu r e 5. Effects of ketoconazole, fluvoxamine, and R-naph-
P450 3A4, 2-hydroxylations of estradiol and estrone
thoflavone on estrone 2-hydroxylation (A), 4-hydroxylation (B),
catalyzed by liver microsomes (sample HL-C19) were
significantly suppressed (but not enhanced) by R-naph-
thoflavone as well as fluvoxamine (Figure 6). In contrast,
16R-hydroxylation of estrone was not affected signifi-
cantly by the addition of fluvoxamine or R-naphtho-
flavone.
and 16R-hydroxylation (C) catalyzed by human liver microsomal
samples HL-C6 and HL-C19. Control activities (nmol of products/
min/nmol of microsomal P450) of 2-, 4-, and 16R-hydroxylations
of estrone in the absence of chemicals were 0.21, 0.015, and
0.043 for liver microsomal sample HL-C6 and 0.51, 0.018, and
0.11 for sample HL-C19, respectively.
nificant inhibition of the 2-, 4-, and 16R-hydroxylations
of estradiol, and these reactions were all found to be
enhanced by R-naphthoflavone (Figure 4). Estradiol 16R-
hydroxylation, but not estradiol 2- and 4-hydroxylations,
was inhibited by the P450 1A2 inhibitor fluvoxamine in
HL-C6 liver microsomes. However, when liver microso-
mal sample HL-C19 (which had significant amounts of
P450s 1A2, 2C9, and 3A4) was used, fluvoxamine sig-
nificantly suppressed the estradiol hydroxylations, being
more inhibitory than ketoconazole. In addition, R-naph-
thoflavone enhanced the 4- and 16R-hydroxylations, but
not 2-hydroxylation, of estradiol by sample HL-C19 liver
microsomes. Sulfaphenazole inhibited estradiol 4-hy-
droxylation catalyzed by human sample HL-C19 mi-
crosomes by 40-50%.
Effects of chemical P450 modifiers on estrone hydroxy-
lations catalyzed by liver microsomal samples HL-C6 and
HL-C19 were also determined (Figure 5). Fluvoxamine
inhibited estrone 2- and 4-hydroxylations while 16R-
hydroxylation of estrone was not affected significantly
by fluvoxamine in human liver sample HL-C6 mi-
crosomes. In contrast, ketoconazole was very effective
in inhibiting estrone 16R-hydroxylation by liver sample
HL-C6 and HL-C19 microsomes, while it did not affect
Discu ssion
Recent studies have established that both P450s 1A2
and 3A4 are able to catalyze oxidations of estradiol and
estrone in human liver microsomes and that other P450
enzymes such as P450s 2C9 and 2E1 may have some role
in estrogen catabolism (10, 17-20). It is, however, not
precisely defined which P450 forms are actually involved
in the estradiol and estrone hydroxylations at different
positions in human livers. For example, although
Guengerich et al. (9) first reported that P450 3A4
(P450NF) is involved in 2- and 4-hydroxylations of estra-
diol by human liver microsomes, Ball et al. (10) reported
that the major proteins involved in estradiol metabolism
are in the P450 1A gene family and that members of the
P450 2C and P450 2E gene subfamilies also have
important roles. The results of Kerlan et al. (19) support
the view that 2- and 4-hydroxylations of estradiol are
catalyzed mainly by P450 3A enzymes in humans. There
are also complicated results reporting that several human
P450 enzymes are able to catalyze 16R-hydroxylation of
estrogens in human liver microsomes, although definitive
studies indicating which P450 enzymes are primarily

Oxidation of Estradiol and Estrone by Human P450s
Chem. Res. Toxicol., Vol. 11, No. 6, 1998 663
Ta ble 2. Effects of Ketocon a zole a n d r-Na p h th ofla von e on Estr a d iol Hyd r oxyla tion Activities by Hu m a n Liver
Micr osom a l Sa m p le HL-C6 in th e P r esen ce of An ti-P 450 1A2 IgGa
system
estradiol hydroxylation activities
(nmol of products/min/nmol of P450)
anti-P450 1A2
(5 mg of IgG/nmol of P450)
ketoconazole
R-naphthoflavone
(µM)
(µM)
2-hydroxylation
4-hydroxylation
16R-hydroxylation
-
+
+
+
+
+
-
-
-
-
0.52 ( 0.06
0.17 ( 0.02
0.003 ( 0.002
<0.001
0.21 ( 0.04
0.42 ( 0.10
0.041 ( 0.005
0.025 ( 0.010
0.002 ( 0.001
<0.001
0.027 ( 0.003
0.148 ( 0.020
0.082 ( 0.011
0.065 ( 0.008
0.021 ( 0.011
0.018 ( 0.009
0.071 ( 0.007
0.121 ( 0.020
10
100
-
-
-
10
100
-
a
Results are presented as means ( SD of triplicate determinations.
to the oxidations of estradiol and estrone at 2-, 4-, and
16R-positions in humans and that the roles of these P450
enzymes vary with the use of human samples used and
with the positions of estrogen hydroxylations determined.
The results obtained in this study can be summarized
as follows.
First, estradiol and estrone 2-hydroxylations are mainly
catalyzed by P450 1A2 in human livers because anti-P450
1A2 and fluvoxamine inhibited these reactions signifi-
cantly and recombinant P450 1A2 had a higher Vmax/Km
ratio than P450 3A4. However, minor but significant
roles of P450 3A4 were found in the 2-hydroxylation of
estradiol and estrone by human livers, since (in human
samples which contained high levels of P450 3A4) both
reactions were inhibited by anti-P450 3A4 and ketocona-
zole and were stimulated by R-naphthoflavone, a chemi-
cal known to enhance some P450 3A4-dependent xeno-
biotic oxidations (45, 46).
F igu r e 6. Effects of fluvoxamine and R-naphthoflavone (both
50 µM) on estradiol 2-hydroxylation (A) and 16R-hydroxylation
(B) and on estrone 2-hydroxylation (C) and 16R-hydroxylation
(D) catalyzed by human liver microsomal sample HL-C19 when
anti-CYP3A4 antibodies (5 mg of IgG/nmol of microsomal P450)
were present in the incubation mixture. Control activities (nmol
of products formed/min/nmol of microsomal P450) in the absence
of antibodies and chemicals were 0.68, 0.09, 0.55, and 0.11 for
2-hydroxylation and 16R-hydroxylation of estradiol and 2-hy-
droxylation and 16R-hydroxylation of estrone, respectively.
Results are presented as means of duplicate determinations.
Second, 4-hydroxylations of estradiol and estrone are
also catalyzed principally by P450 1A2 in human liver
microsomes, but the roles of P450 3A4 in the 4-hydroxy-
lation reactions may be more significant than those seen
in the 2-hydroxylations of estradiol and estrone. For
example, when liver sample HL-C6 (which contained
P450 3A4 at a high level) was used, anti-P450 1A2
inhibited estradiol 4-hydroxylation only ∼40% and the
resultant activities were completely inhibited by keto-
conazole and significantly enhanced by R-naphthoflavone.
Such enhancements of 4-hydroxylation activities by
R-naphthoflavone were also seen when estrone was
incubated with human liver microsome sample HL-C19,
as well as with HL-C6. It should, however, be mentioned
that roles of P450 1A2 in the 4-hydroxylation of estradiol
and estrone may be more significant than those by P450
involved in the 16R-hydroxylation of estradiol and estrone
are lacking (17, 18).
Why have such complicated results been reported from
several laboratories on the roles of P450 enzymes in-
volved in the oxidation of estrogens by human liver
microsomes? It has been shown that there are interin-
dividual variations in the levels of individual P450
enzymes in humans (36-40). For example, average
levels of P450 3A4 in human livers have been determined
to be ∼30% of total P450 in 30 J apanese and 30
Caucasians examined; in some people this P450 level
accounts for >60% of total P450, probably due to the
induction of P450 3A4 by various chemical agents (41-
44). It is also known that P450 1A2 is induced in human
livers by several chemicals such as polycyclic aromatic
hydrocarbons, arylamines, and chlorinated hydrocarbons
through ingestion of charcoal-broiled foods, smoking, and
other exposures (3, 42, 44). These results support the
view that contributions of P450 enzymes in estrogen
hydroxylation reactions in human livers may be altered
by using different human liver samples whose composi-
tions of various P450 enzymes differ.
3A4, because recombinant P450 1A2 had a higher Vmax
/
Km ratio than P450 3A4 and anti-P450 1A2 inhibited
estradiol and estrone 4-hydroxylations more than anti-
P450 3A4 in liver microsome HL-C19 sample, in which
P450 1A2 and 3A4 levels in the liver were estimated to
be 25% and 46%, respectively, of total P450.
Third, estradiol 16R-hydroxylation is suggested to be
catalyzed primarily by both P450s 1A2 and 3A4 in human
liver microsomes, although the roles of these two P450
enzymes in the reaction were affected by which human
samples were used for the determinations. In contrast,
estrone 16R-hydroxylation is found to be catalyzed most
actively by P450 3A4, because recombinant P450 3A4
catalyzed this reaction at the highest level and anti-P450
3A4 was very effective in inhibiting the reaction. Roles
of P450 1A2 in estrone 16R-hydroxylation may be very
minor due to the fact that the recombinant enzyme
catalyzed estrone 16R-hydroxylation at a very low level
Using different human samples that contain varying
levels of individual P450 enzymes in the liver and also
recombinant human P450 enzymes expressed in bacu-
lovirus systems, we obtained several pieces of evidence
to support the view that different human P450 enzymes,
particularly P450s 1A2 and 3A4, contribute significantly

664 Chem. Res. Toxicol., Vol. 11, No. 6, 1998
Yamazaki et al.
(8) Yamazaki, H., and Shimada, T. (1997) Progesterone and test-
osterone hydroxylation by cytochromes P450 2C19, 2C9, and 3A4
in human liver microsomes. Arch. Biochem. Biophys. 346, 161-
169.
and that anti-P450 1A2 poorly inhibited the reaction
catalyzed by human liver microsomes.
P450 2C9 may also make some, but not a major,
contribution to estrogen metabolism. Although recom-
binant P450 2C9 catalyzed oxidations of estradiol and
estrone at several positions, anti-P450 2C9 and sul-
faphenazole did not significantly affect estradiol and
estrone hydroxylations catalyzed by human liver mi-
crosomes. Other P450 enzymes including P450s 2B6,
2D6, and 2E1 are also suggested to have only minor roles
in estrogen catabolism in humans, since the recombinant
enzymes had very low catalytic activities for the hydroxy-
lation reactions.
(9) Guengerich, F. P., Martin, M. V., Beaune, P. H., Kremers, P.,
Wolff, T., and Waxman, D. J . (1986) Characterization of rat and
human liver microsomal cytochrome P-450 forms involved in
nifedipine oxidation, a prototype for genetic polymorphism in
oxidative drug metabolism. J . Biol. Chem. 261, 5051-5060.
(10) Ball, S. E., Forrester, L. M., Wolf, C. R., and Back, D. J . (1990)
Differences in the cytochrome P-450 isoenzymes involved in the
2-hydroxylation of oestradiol and 17R-ethinyloestradiol. Relative
activities of rat and human liver enzymes. Biochem. J . 267, 221-
226.
(11) Yager, J . D., and Liehr, J . G. (1996) Molecular mechanisms of
estrogen carcinogenesis. Annu. Rev. Pharmacol. Toxicol. 36, 203-
232.
In conclusion, our present results suggested that both
P450s 1A2 and 3A4 are major P450 enzymes involved in
the oxidations of estrogens in human livers. The roles
of these two P450s vary depending on the contents of
P450s 1A2 and 3A4 in livers. In general, P450 1A2 may
be more active in catalyzing 2- and 4-hydroxylations of
estradiol and estrone than P450 3A4, while the latter
enzyme catalyzes the 16R-hydroxylation of estrone more
than P450 1A2. Estradiol 16R-hydroxylation is catalyzed
by several P450 enzymes including P450s 1A2 and 3A4.
This information is important because human P450 1A2
is expressed only in liver but P450 3A4 is also expressed,
at lower concentrations, in a number of extrahepatic
tissues (2). Therefore, the P450 3A4-catalyzed hydroxy-
lations may have special significance at these sites. P450
2C enzymes may make some contribution. This informa-
tion about the roles of individual human P450s in
estrogen 2-, 4-, and 16R-hydroxylations is of relevance
in adhering current controversies of hormonal estrogen
and “environmental estrogen” carcinogenesis (47, 48).
(12) Lemon, H. M., Heidel, J . W., and Rodriguez-Sierra, J . F. (1992)
Increased catechol estrogen metabolism as
a risk factor for
nonfamilial breast cancer. Cancer 69, 457-465.
(13) Liehr, J . G., and Rocci, M. J . (1996) 4-Hydroxylation of estrogens
as marker of human mammary tumors. Proc. Natl. Acad. Sci.
U.S.A. 93, 3294-3296.
(14) Nebert, D. W. (1993) Elevated estrogen 16R-hydroxylase activ-
ity: Is this a genotoxic or nongenotoxic biomarker in human
breast cancer risk? J . Natl. Cancer Inst. 85, 1888-1891.
(15) Bradlow, H. L., Hershcopf, R. J ., Martucci, C. P., and Fishman,
J . (1985) Estradiol 16R-hydroxylation in the mouse correlates with
mammary tumor incidence and presence of murine mammary
tumor virus: a possible model for the hormonal etiology of breast
cancer in humans. Proc. Natl. Acad. Sci. U.S.A. 82, 6295-6299.
(16) Osborne, M. P., Bradlow, H. L., Wong, G. Y. C., and Telang, N.
T. (1993) Upregulation of estradiol C16R-hydroxylation in human
breast tissue: a potential biomarker of breast cancer risk. J . Natl.
Cancer Inst. 85, 1917-1920.
(17) Aoyama, T., Korzekwa, K., Nagata, K., Gillette, J ., Gelboin, H.
V., and Gonzalez, F. J . (1990) Estradiol metabolism by comple-
mentary deoxyribonucleic acid-expressed human cytochrome
P450s. Endocrinology 126, 3101-3106.
(18) Shou, M., Korzekwa, K. R., Brooks, E. N., Krausz, K. W.,
Gonzalez, F. J ., and Gelboin, H. V. (1997) Role of human hepatic
cytochrome P450 1A2 and 3A4 in the metabolic activation of
estrone. Carcinogenesis 18, 207-214.
Ack n ow led gm en t. This work was supported in part
by grants from the Ministry of Education, Science, and
Culture of J apan, the Ministry of Health and Welfare of
J apan, and the Developmental and Creative Studies from
Osaka Prefectural Government and also by United States
Public Health Service Grants R35 CA44353 and P30
ES00267.
(19) Kerlan, V., Dreano, Y., Bercovici, J . P., Beaune, P. H., Floch, H.
H., and Berthou, F. (1992) Nature of cytochrome P450 involved
in the 2-/4-hydroxylations of estradiol in human liver microsomes.
Biochem. Pharmacol. 44, 1745-1756.
(20) Hayes, C. L., Spink, D. C., Spink, B. C., Cao, J . Q., Walker, N. J .,
and Sutter, T. R. (1996) 17â-Estradiol hydroxylation catalyzed
by human cytochrome P450 1B1. Proc. Natl. Acad. Sci. U.S.A.
93, 9776-9781.
(21) Spink, D. C., Eugster, H. P., Lincoln, D. W., II, Schuetz, J . D.,
Schuetz, E. G., J ohnson, J . A., Kaminsky, L. S., and Gierthy, J .
F. (1992) 17â-Estradiol hydroxylation catalyzed by human cyto-
chrome P450 1A1: a comparison of the activities induced by
2,3,7,8-tetrachlorodibenzo-p-dioxin in MCF-7 cells with those from
heterologous expression of the cDNA. Arch. Biochem. Biophys.
293, 342-348.
(22) Yamazaki, H., Gillam, E. M. J ., Dong, M.-S., J ohnson, W. W.,
Guengerich, F. P., and Shimada, T. (1997) Reconstitution of
recombinant cytochrome P450 2C10 (2C9) and comparison with
cytochrome P450 3A4 and other forms: Effects of cytochrome
P450-P450 and cytochrome P450-b5 interactions. Arch. Biochem.
Biophys. 342, 329-337.
(23) Yamazaki, H., and Shimada, T. (1997) Human liver cytochrome
P450 enzymes involved in the 7-hydroxylation of R- and S-
warfarin enantiomers. Biochem. Pharmacol. 54, 1195-1203.
(24) Shaw, P. M., Hosea, N. A., Thompson, D. V., Lenius, J . M., and
Guengerich, F. P. (1997) Reconstitution premixes for assays using
purified recombinant human cytochrome P450, NADPH-cyto-
chrome P450 reductase, and cytochrome b5. Arch. Biochem.
Biophys. 348, 107-115.
Refer en ces
(1) Nelson, D. R., Koymans, L., Kamataki, T., Stegeman, J . J .,
Feyereisen, R., Waxman, D. J ., Waterman, M. R., Gotoh, O., Coon,
M. J ., Estabrook, R. W., Gunsalus, I. C., and Nebert, D. W. (1996)
P450 superfamily: update on new sequences, gene mapping,
accession numbers and nomenclature. Pharmacogenetics 6, 1-42.
(2) Guengerich, F. P. (1995) Human cytochrome P450 enzymes. In
Cytochrome P450 (Oritiz de Montellano, P. R., Ed.) pp 473-535,
Plenum Press, New York.
(3) Guengerich, F. P., and Shimada, T. (1991) Oxidation of toxic and
carcinogenic chemicals by human cytochrome P-450 enzymes.
Chem. Res. Toxicol. 4, 391-407.
(4) Gustafsson, J .-A., Mode, A., Norstedt, G., and Skett, P. (1983)
Sex steroid induced changes in hepatic enzymes. Annu. Rev.
Physiol. 45, 51-60.
(5) Ryan, D. E., and Levin, W. (1990) Purification and characteriza-
tion of hepatic microsomal cytochrome P-450. Pharmacol. Ther.
45, 153-239.
(6) Swinney, D. C. (1990) Progesterone metabolism in hepatic mi-
crosomes. Effect of the cytochrome P-450 inhibitor, ketoconazole,
and the NADPH 5R-reductase inhibitor, 4-MA, upon the metabolic
profile in human, monkey, dog, and rat. Drug Metab. Dispos. 18,
859-865.
(7) Waxman, D. J ., Attisana, C., Guengerich, F. P., and Lapenson,
D. P. (1988) Human liver microsomal steroid metabolism: iden-
tification of the major microsomal steroid hormone 6â-hydroxylase
cytochrome P-450 enzyme. Arch. Biochem. Biophys. 263, 424-
436.
(25) Yamazaki, H., Inoue, K., Shaw, P. M., Checovich, W. J ., Guenger-
ich, F. P., and Shimada, T. (1997) Different contributions of
cytochrome P450 2C19 and 3A4 in the oxidation of omeprazole
by human liver microsomes: Effects of contents of these two forms
in individual human samples. J . Pharmacol. Exp. Ther. 283, 434-
442.
(26) Yamazaki, H., Inoue, K., Turvy, C. G., Guengerich, F. P., and
Shimada, T. (1997) Effects of freezing, thawing, and storage of
human liver samples on the microsomal contents and activities
of cytochrome P450 enzymes. Drug Metab. Dispos. 25, 168-174.

Oxidation of Estradiol and Estrone by Human P450s
Chem. Res. Toxicol., Vol. 11, No. 6, 1998 665
(27) Inoue, K., Yamazaki, H., Imiya, K., Akasaka, S., Guengerich, F.
P., and Shimada, T. (1997) Relationship between CYP2C9 and
2C19 genotypes and tolbutamide methyl hydroxylation and
S-mephenytoin 4′-hydroxylation activities in livers of J apanese
and Caucasian populations. Pharmacogenetics 7, 103-113.
(28) Guengerich, F. P. (1994) Analysis and characterization of en-
zymes. In Principles and Methods of Toxicology (Hayes, A. W.,
Ed.) pp 1259-1313, Raven Press, New York.
(29) Distlerath, L. M., Reilly, P., Martin, M. V., Davis, G. G.,
Wilkinson, G. R., and Guengerich, F. P. (1985) Purification and
characterization of the human liver cytochromes P-450 involved
in debrisoquine 4-hydroxylation and phenacetin O-deethylation,
two prototypes for genetic polymorphism in oxidative drug
metabolism. J . Biol. Chem. 260, 9057-9067.
(30) Shimada, T., Misono, K. S., and Guengerich, F. P. (1986) Human
liver microsomal cytochrome P-450 mephenytoin 4-hydroxylase,
a prototype of genetic polymorphism in oxidative drug metabo-
lism. Purification and characterization of two similar forms
involved in the reaction. J . Biol. Chem. 261, 909-921.
(31) Kaminsky, L. S., Fasco, M. J ., and Guengerich, F. P. (1981)
Production and application of antibodies to rat liver cytochrome
P-450. Methods Enzymol. 74, 262-272.
(38) Imaoka, S., Yamada, T., Hiroi, T., Hayashi, K., Sakai, T.,
Yabusaki, Y., and Funae, Y. (1996) Multiple forms of human P450
expressed in Saccharomyces cerevisiae. Systematic characteriza-
tion and comparison with those of the rat. Biochem. Pharmacol.
51, 1041-1050.
(39) Lucas, D., Berthou, F., Dreano, Y., Lozac′h, P., Volant, A., and
Menez, J . F. (1993) Comparison of levels of cytochromes P-450,
CYP1A2, CYP2E1, and their related monooxygenase activities in
human surgical liver samples. Alcohol Clin. Exp. Res. 17, 900-
905.
(40) Forrester, L. M., Henderson, C. J ., Glancey, M. J ., Back, D. J .,
Park, B. K., Ball, S. E., Kitteringham, N. R., McLaren, A. W.,
Miles, J . S., Skett, P., and Wolf, C. R. (1992) Relative expression
of cytochrome P450 isoenzymes in human liver and association
with the metabolism of drugs and xenobiotics. Biochem. J . 281,
359-368.
(41) Renidic, S., and Di Carlo, F. J . (1997) Human cytochrome P450
enzymes: A status report summarizing their reactions, sub-
strates, inducers, and inhibitors. Drug Metab. Rev. 29, 413-580.
(42) Wrighton, S. A., and Stevens, J . C. (1992) The human hepatic
cytochromes P450 involved in drug metabolism. Crit. Rev. Toxicol.
22, 1-21.
(32) Shimada, T., and Nakamura, S. (1987) Cytochrome P-450-
mediated activation of procarcinogens and promutagens to DNA-
damaging products by measuring expression of umu gene in
Salmonella typhimurium TA1535/pSK1002. Biochem. Pharmacol.
36, 1979-1987.
(33) Omura, T., and Sato, R. (1964) The carbon monoxide-binding
pigment of liver microsomes. I. Evidence for its hemoprotein
nature. J . Biol. Chem. 239, 2370-2378.
(34) Lowry, O. H., Rosebrough, N. J ., Farr, A. L., and Randall, R. J .
(1951) Protein measurement with the Folin phenol reagent. J .
Biol. Chem. 193, 265-275.
(35) Guengerich, F. P., Wang, P., and Davidson, N. K. (1982) Estima-
tion of isozymes of microsomal cytochrome P-450 in rats, rabbits,
and humans using immunochemical staining coupled with sodium
dodecyl sulfate-polyacrylamide gel electrophoresis. Biochemistry
21, 1698-1706.
(36) Shimada, T., Yamazaki, H., Mimura, M., Inui, Y., and Guengerich,
F. P. (1994) Interindividual variations in human liver cytochrome
P-450 enzymes involved in the oxidation of drugs, carcinogens
and toxic chemicals: studies with liver microsomes of 30 J apanese
and 30 Caucasians. J . Pharmacol. Exp. Ther. 270, 414-423.
(37) Transon, C., Lecoeur, S., Leemann, T., Beaune, P., and Dayer, P.
(1996) Interindividual variability in catalytic activity and immu-
noreactivity of three major human liver cytochrome P450 isozymes.
Eur. J . Clin. Pharmacol. 51, 79-85.
(43) Pichard, L., Fabre, I., Daujat, M., Domergue, J ., J oyeux, H., and
Maurel, P. (1992) Effect of corticosteroids on the expression of
cytochromes P450 and on cyclosporin a oxidase activity in primary
cultures of human hepatocytes. Mol. Pharmacol. 41, 1047-1055.
(44) Guengerich, F. P. (1989) Characterization of human microsomal
cytochrome P-450 enzymes. Annu. Rev. Pharmacol. Toxicol. 29,
241-264.
(45) Shimada, T., and Okuda, Y. (1988) Metabolic activation of
environmental carcinogens and mutagens by human liver micro-
somes: role of cytochrome P-450 homologous to a 3-methylcholan-
threne-inducible isozyme in rat liver. Biochem. Pharmacol. 37,
459-465.
(46) Shimada, T., Iwasaki, M., Martin, M. V., and Guengerich, F. P.
(1989) Human liver microsomal cytochrome P-450 enzymes
involved in the bioactivation of procarcinogens detected by umu
gene response in Salmonella typhimurium TA1535/pSK1002.
Cancer Res. 49, 3218-3228.
(47) Bradlow, H. L., Davis, D. L., Lin, G., Sepkovic, D., and Tiwari,
R. (1995) Effects of pesticides on the ratio of 16R/2-hydroxy-
estrone: a biologic marker of breast cancer risk. Environ. Health
Perspect. 103, 147-150.
(48) Service, R. F. (1998) New role for estrogen in cancer? Science 279,
1631-1633.
TX970217F