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Biochemistry and Biophysics |
Departments of Biochemistry [I. H. H., F. P. G.] and Pathology[S. D., N. R., F. F. P.], Vanderbilt University Medical Center, Nashville, Tennessee 37232
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ABSTRACT |
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hydroxylation product, 16-OH-E2, has been postulated to be a factor
in mammary carcinogenesis. Cytochrome P450 1B1 (CYP1B1) exceeds other
P450 enzymes in both estrogen hydroxylation activity and expression
level in breast tissue. To determine whether inherited variants of
CYP1B1 differ from wild-type CYP1B1 in estrogen hydroxylase activity,
we expressed recombinant wild-type and five polymorphic variants of
CYP1B1: variant 1 (codon 48Arg
Gly), variant 2 (codon 119AlaSer),
variant 3 (codon 432ValLeu), variant 4 (codon453Asn Ser), variant
5 (48Gly, 119Ser, 432Leu, 453Ser). The His-tagged proteins were
purified by nickel-nitrilotriacetic acid (Ni-NTA) chromatography
and analyzed by electrophoresis and spectrophotometry. We performed
assays of E2 hydroxylation activity and quantitated production of
2-OH-E2, 4-OH-E2, and 16-OH-E2 by gas chromatography/mass
spectrometry. Wild-type CYP1B1 formed 4-OH-E2 as main product
(Km, 40 ± 8
µM; kcat 4.4 ± 0.4, min-1;
kcat/Km, 110
mM-1min-1), followed by 2-OH-E2
(Km, 34 ± 4
µM; kcat, 1.9 ± 0.1 min-1;
kcat/Km, 55
mM-1min-1) and 16-OH-E2
(Km, 39 ± 5.7
µM; kcat, 0.30 ± 0.02 min-1;
kcat/Km, 7.6
mM-1min-1). The CYP1B1 variants
also formed 4-OH-E2 as the main product but displayed 2.4- to 3.4-fold
higher catalytic efficiencies
kcat/Km than the
wild-type enzyme, ranging from 270
mM-1min-1 for variant 4, to 370
mM-1min-1 for variant 2. The
variant enzymes also exceeded wild-type CYP1B1 with respect to 2- and
16-hydroxylation activity. Thus, inherited alterations in CYP1B1
estrogen hydroxylation activity may be associated with significant
changes in estrogen metabolism and, thereby, may possibly explain
interindividual differences in breast cancer risk associated with
estrogen-mediated carcinogenicity. |
INTRODUCTION |
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hydroxylation. Two enzymes,
CYP1A1 and CYP1B1, are responsible for the hydroxylation to the 2-OH
and 4-OH catechol estrogens (i.e., 2-OH-E2 and 4-OH-E2). The
2-OH and 4-OH catechol estrogens are oxidized to semiquinones and
quinones. The latter are reactive electrophilic metabolites and are
capable of forming DNA adducts (1
, 2)
. Further DNA damage
results from quinone-semiquinone redox cycling, generated by enzymatic
reduction of catechol estrogen quinones to semiquinones and subsequent
auto-oxidation back to quinones (3, 4, 5, 6)
. C-16
hydroxylation has also been suggested to be involved in breast
carcinogenesis (7
, 8) . Although other cytochrome P450 enzymes, such as CYP1A2 and CYP3A4, are involved in hepatic and extrahepatic estrogen hydroxylation, CYP1A1 and CYP1B1 display the highest levels of expression in breast tissue (9 , 10) . In turn, CYP1B1 exceeds CYP1A1 in its catalytic efficiency as an E2 hydroxylase and differs from CYP1A1 in its principal site of catalysis (11, 12, 13) . CYP1B1 has its primary activity at the C-4 position of E2, whereas CYP1A1 has its primary activity at the C-2 position in preference to 4-hydroxylation. The 4-hydroxylation activity of CYP1B1 has received particular attention because of the fact that the 2-OH and 4-OH catechol estrogens differ in carcinogenicity. Treatment with 4-OH-E2, but not 2-OH-E2, induced renal cancer in Syrian hamster (14 , 15) . Analysis of renal DNA demonstrated that 4-OH-E2 significantly increased 8-hydroxydeoxyguanosine levels, whereas 2-OH-E2 did not cause oxidative DNA damage (16) . Similarly, 4-OH-E2 induced DNA single-strand breaks whereas 2-OH-E2 had a negligible effect (17) . Comparison of the corresponding catechol estrogen quinones showed that E2–3,4quinone produced two to three orders of magnitude higher levels of depurinating adducts than E2–2,3-quinone (18) . In addition to the induction of renal cancer in the hamster model, 4-OH-E2 is capable of inducing uterine adenocarcinoma, a hormonally related cancer, in mice. Administration of E2, 2-OH-E2, and 4-OH-E2 induced endometrial carcinomas in 7, 12, and 66%, respectively, of treated CD-1 mice (19) . Finally, examination of microsomal E2 hydroxylation in human breast cancer showed significantly higher 4-OH-E2/2-OH-E2 ratios in tumor tissue than in adjacent normal breast tissue (20) . All of these findings support a causative role of 4-OH catechol estrogens in carcinogenesis and implicate CYP1B1 as a key player in the process.
Mutations and polymorphisms have both been identified in the
CYP1B1 gene. Primary congenital glaucoma, a rare autosomal
recessive eye disorder, has been linked to homozygous frameshift and
missense mutations in affected Turkish and Saudi Arabian families
(21, 22, 23)
. Six polymorphisms of the CYP1B1 gene
have been described in the Anglo-American population, of which four
result in amino acid substitutions (Table 1
; Refs. 23
, 24
). We described two of these amino acid substitutions in exon
3, which encodes the heme-binding domain: codon 432ValLeu and codon
453AsnSer (24)
. Stoilov et al.
(23)
described the other two amino acid substitutions in
codons 48ArgGly and 119AlaSer in exon 2. Polymorphisms are
inherited and, therefore, dictate exposure levels to metabolites for
life. Thus, inherited alterations in the activity of CYP1B1 hold the
potential to define differences in estrogen metabolism and, thereby,
possibly explain interindividual differences in breast cancer risk
associated with estrogen-mediated carcinogenesis. However, to support
this hypothesis, formal proof is needed that these inherited enzyme
variants are indeed associated with significant changes in estrogen
metabolism. In the present study, we determined whether the polymorphic
variants of CYP1B1 differ from wild-type CYP1B1 in 2-, 4-, and
16-estradiol hydroxylation activities.
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MATERIALS AND METHODS |
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F'Iq using the methods described by the
manufacturer. Colonies harboring the correct sequence (as judged by
restriction digest and DNA sequencing) were picked and used to express
the respective CYP1B1 protein.
Expression and Purification of Recombinant CYP1B1.
Recombinant wild-type and variant CYP1B1 proteins were expressed in
Escherichia coli. Strain DH5F'Iq yielded the highest
expression levels. Transformed DH5F'Iq cells were grown for 12 h at 37°C in 50 ml of modified terrific broth medium
containing 100 µg of ampicillin/ml, 25 µg of kanamycin/ml, 1
mM thiamine, and 10 mM
glucose. The cells were then grown at 33°C in the same medium with
added trace elements as described previously (25)
until
the A600 nm was between 0.6
and 0.9. Mild induction with 8 mM lactose yielded
optimal enzyme production if 0.5 mM
-aminolevulinic acid was added and cells were grown at 23°C for
40 h while shaking at 150 rpm. After 40 h, cells were
harvested by centrifugation at 6,500 x g for
10 min, and the P450 content in the bacterial cell lysate was
determined by Fe2+-CO versus
Fe2+ difference spectra. Spheroplasts were
prepared with the use of lysozyme and disrupted by sonication. The
pellet obtained after centrifugation at 10,000 x g for 20 min was discarded, and the microsomal membranes in
the supernatant was used as a source for purification. The membranes
were pelleted by overnight centrifugation at 110,000 x g, and the resultant supernatant was discarded because it
generally contained <3% of the P450 content. The red 110 K
pellet was resuspended in 200 ml of solubilization buffer [100
mM NaPO4 (pH 8.0), 0.4
M NaCl, 40% glycerol (v/v), 10
mM ß-mercaptoethanol, 10
µM aprotinin, 0.5% sodium cholate (w/v), and
1.0% Triton N-101 (w/v)], and the suspension was stirred overnight.
Centrifugation at 110,000 x g for 90 min
yielded a clear pellet, which was discarded, and a supernatant that
contained most of the P450. The supernatant was applied to a
preequilibrated Ni-NTA column (1 ml of resin per 50 nmol of
enzyme). The column was washed with at least 50 column volumes of wash
buffer [100 mM NaPO4 (pH
8.0), 0.4 M NaCl, 40% glycerol (v/v), 10
mM ß-mercaptoethanol, 0.25% sodium cholate
(w/v), and 10 mM imidazole], followed by a
second wash with the same buffer containing 40 mM
imidazole to remove unbound proteins and Triton N-101. The His-tagged
protein was eluted with two column volumes of buffer [100
mM NaPO4 (pH 8.0), 0.4
M NaCl, 40% glycerol (v/v), 10
mM ß-mercaptoethanol, 0.25% sodium cholate
(w/v), and 400 mM imidazole], and the eluate
dialyzed against dialysis buffer [100 mM
NaPO4 (pH 7.4), 0.25 M
NaCl, 1 mM EDTA, 20% glycerol (v/v), and 0.1
mM DTT). The purity of the protein was assessed
by SDS-PAGE and silver staining and by Western immunoblots using both
anti-(oligo)His and anti-CYP1B1 antibodies.
Site-Directed Mutagenesis.
Part of our initial studies of the CYP1B1 gene, including
DNA sequence analysis, was carried out with human breast cancer cell
lines. In analyzing the CYP1B1 gene in cell lines, we
determined that BT-20 cells contain the CYP1B1 sequence designated as
wild type. Accordingly, CYP1B1 cDNA from BT-20 cells served as the
source for site-directed mutagenesis and the corresponding pQE-30
wild-type CYP1B1 plasmid was used as template to generate variant
CYP1B1 cDNA encoding the substitutions in codon 48, 119, 432, and 453
(Table 1)
. Complementary 25 base oligonucleotide primers were synthesized to
contain the selected mutated nucleotides in the center and were
purified by PAGE. We used the primers in the QuikChange site-directed
mutagenesis method as specified by the manufacturer (Stratagene). After
12 PCR cycles with TurboPfu DNA polymerase, the reaction was
digested with DpnI and transformed into XL1-Blue cells.
Successful mutagenesis was verified by nucleotide sequence analysis.
Transformation into DH5F'Iq cells, expression, and purification of
variant CYP1B1 were performed as described above.
Spectrophotometric Analyses.
All of the spectra were recorded using an Aminco DW2a/Olis
instrument (On-Line Instrument Systems, Bogart, GA). Wavelength maxima
were determined using the peak finder or second derivative software.
The high-spin content was estimated from the second derivative spectrum
of the ferric enzyme as described previously (26)
. P450
and cytochrome P420 concentrations were determined as described
previously (27)
.
Assay of CYP1B1 E2 Hydroxylation Activity.
Purified CYP1B1 (200 pmol) was reconstituted with a 2-fold molar amount
of recombinant rat NADPH-P450 reductase (400 pmol), purified as
described previously (28)
, and with 60 µg of
L--dilauroyl-sn-glycero-3-phosphocholine in
the presence of sodium cholate (0.005%, w/v; Ref. 29
) in
0.4 ml of 100 mM potassium phosphate buffer (pH
7.4) containing varying concentrations of E2 (2, 3, 6, 9, 12, 15, 20,
40, 60, 80, and 100 µM) and 1
mM ascorbate. A NADPH-generating system
consisting of 5 mM glucose 6-phosphate and 0.5
units of glucose-6-phosphate dehydrogenase/ml was added and reactions
were initiated by adding NADP+ to a final
concentration of 0.5 mM. Reactions proceeded for
10 min at 37°C with gentle shaking and then were terminated by the
addition of 2 ml of CH2Cl2.
Extraction and Gas Chromatography/Mass Spectrometry Analysis of
E2 and Metabolites.
A deuterated internal standard (100 µl of 8 mg/liter E2–2, 4,
16, 16-d4 in methanol; CDN Isotopes,
Pointe-Claire, Quebec, Canada) was added, and all of the steroids were
extracted into CH2Cl2 by
vortex mixing for 30 s. The
CH2Cl2 fraction (1.5 ml)
was evaporated to dryness under air and volatile TMS derivatives
were prepared by heating the residue with 100 µl of 50%
NO-bis(trimethylsilyl)trifluoroacetamide/1% trimethyl chlorosilane in
acetonitrile at 56°C for 30 min. The TMS derivatives of E2 and its
metabolites were separated by gas chromatography (H-P 5890,
Hewlett-Packard, Wilmington, DE) on a 5% phenyl methyl silicone
stationary-phase fused silica capillary column (30 m x 0.2 mm x 0.5 µm film, HP5; Hewlett-Packard). Helium
carrier gas was used at a flow of 1 ml/min. The injector was operated
at 250°C, with 2 µl injected in the splitless mode, with a purge
(60 ml/min helium) time of 0.6 min. The oven temperature was held at
180°C for 0.5 min, then raised at 6°C/min to 250°C, at which it
was held for 17 min, then raised to 300°C at 8°C/min to give a
total run time of 35.42 min. This program permitted adequate separation
of a wide range of estrogen metabolites. Retention times for the TMS
derivatives were: E2 and E2-d4, 20.6 min;
2-OH-E2, 26.6 min; 4-OH-E2, 28.7 min; and 16-OH-E2, 30.3 min. The
mass spectrometer (H-P 5970) was operated in the electron impact
selected ion monitoring mode from 18 to 34 min. Ions monitored were
TMS2-E2-d4 420, 288, 330;
TMS2-E2 416, 285, 326;
TMS3-2-OH-E2 504, 373;
TMS3-4-OH-E2 504, 373, 325;
TMS3-16-OH-E2 345, 311, 504. The instrument
was calibrated by simultaneous preparation of an 11-point calibration
over the range 0- 10.5 nmol/tube of each compound. Sensitivity was
determined to be between 0.02 and 0.04 nmol/tube (400- 800 fmol on
column) for the various compounds. Preparation of the TMS derivatives
improved chromatography and sensitivity significantly. Derivatization
was performed at 56°C because the use of a higher temperature
resulted in the loss of some estrogen derivatives (particularly the
2-OH metabolite of estrone). Derivatization was demonstrated to be
complete at 20 min as evidenced by the absence of detectable amounts of
underivatized estrogens in the highest calibrator when the detector was
operated in full scan mode. Absolute extraction efficiency for E2,
2-OH-E2, and 4-OH-E2 at 3.5 nmol/tube was 119, 96, and 107%,
respectively, assessed by comparison with injections of spiked solvent
samples onto the gas chromatograph. The internal standard added before
extraction compensated for deviation from 100% recovery.
Statistical Analysis.
Kinetic parameters (Km and
kcat) were determined by
nonlinear regression analysis using the computer program GraphPad PRISM
(San Diego, CA).
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RESULTS |
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F'Iq instead of strains recommended by the
manufacturer (Qiagen) and the induction of protein expression with
lactose instead of
isopropyl-ß-D-thiogalactopyranoside. The
protein modification strategy (i.e., replacement of the
NH2-terminal hydrophobic segment) did not affect
the intracellular localization of the recombinant protein in bacterial
membranes. However, a much longer centrifugation period was required in
the 110,000 x g sedimentation step to pellet
the majority of the expressed protein. The presence of the
NH2-terminal hexahistidine allowed purification
of the recombinant proteins with relatively high yields (results not
presented). Purified wild-type and variant CYP1B1 were
electrophoretically homogeneous as judged by SDS-PAGE and silver
staining, which revealed a single band at
Mr 55,000 for all of the
proteins (Fig. 1)
. Western immunoblots using both anti-(oligo)His and anti-CYP1B1
antibodies also yielded one major band at
Mr 55,000 (results not shown).
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max at 450 nm and negligible amounts of
cytochrome P420, the denatured form of the enzyme (Fig. 2)
. Examination of the absolute spectra of CYP1B1 revealed that the
ferric protein was nearly all in the low-spin state. The low-spin
character was further verified by examination of the second derivative
spectrum (Fig. 2)
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. Sodium cholate (0.005% w/v) was included in the
reconstitution mixtures as suggested by Shimada et al.
(29)
. However, the exclusion of sodium cholate in separate
experiments did not significantly affect the observed catalytic
properties. The reaction kinetics were determined for each enzyme in
duplicate at ten different concentrations of E2 (Fig. 3)
, and the resulting Km and
kcat values are presented in Table 2
. Wild-type CYP1B1 formed 4-OH-E2 as main product
(Km, 40 ± 8
µM; kcat,
4.4 ± 0.4 min-1;
kcat/Km,
110
mM-1min-1),
followed by 2-OH-E2 (Km, 34 ± 4 µM;
kcat, 1.9 ± 0.1
min-1;
kcat/Km,
55
mM-1min-1)
and 16-OH-E2 (Km, 39.4 ± 5.7 µM;
kcat, 0.30 ± 0.02
min-1;
kcat/Km
7.6
mM-1min-1).
The CYP1B1 variants also formed 4-OH-E2 as main product but
displayed 2.4- to 3.4-fold higher catalytic efficiencies
kcat/Km
than the wild-type enzyme, ranging from 270
mM-1min-1
for variant 4, to 370
mM-1min-1
for variant 2 (Table 2)
. The variant enzymes also exceeded wild-type
CYP1B1 with respect to 2- and 16-hydroxylation activity, although
the differences were smaller (Table 2)
. Overall, the 4-hydroxylation
activity of the various enzymes was 2- to 4-fold higher than the
2-hydroxylation activity and 15- to 45-fold higher than the
16-hydroxylation activity.
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DISCUSSION |
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A polymorphism at nucleotide 1347 is silent,
i.e., the amino acid sequence is not affected;
(d) on the basis of the extensive DNA sequence analysis of
the CYP1B1 gene in 100 individuals by Stoilov et
al. (23)
and our own studies of the CYP1B1
gene (24)
, it seems unlikely that polymorphisms other than
those in codons 48, 119, 432, and 453 are present in the coding region,
at least in Caucasians; and (e) multiple sequence alignment
for 22 different members of the cytochrome P450 superfamily has shown
that the four CYP1B1 polymorphisms associated with amino acid
substitutions, i.e., 48ArgGly, 119AlaSer,
432ValLeu, and 453AsnSer, are near conserved regions
(23)
. In consideration of these data, we focused our
analysis on these four polymorphisms.
We modified the recombinant CYP1B1 proteins to allow efficient
expression and purification from the bacterial host. The replacement of
the NH2 terminus, which serves to anchor the
proteins in the microsomal membrane, by an oligo-His region has been
used with several P450s and shown not to affect enzyme activity
(29, 30, 31)
. The spectral properties of the expressed
proteins were indicative of P450s with low-spin characteristics.
Wild-type CYP1B1 has been expressed in yeast and bacteria. Microsomes
from the transformed yeast catalyzed the 2- and 4-hydroxylation of E2
with kcat values of 0.27 and 1.4
min-1, respectively (13)
. The
corresponding turnover numbers for the bacterially expressed enzyme
were 0.13 and 1.4 min-1, respectively
(29)
. The 2- and 4-hydroxylation activities observed for
wild-type CYP1B1 in the present study were higher, possibly
attributable to the use of purified enzymes in an optimized
reconstitution system. However, all of the studies are in agreement
that CYP1B1 preferentially catalyzes E2 4-hydroxylation, although small
modifications have been made to facilitate expression. Comparison with
other P450 enzymes has shown that the catalytic efficiency of CYP1B1
for 4-hydroxylation was greater than the catalytic efficiencies of
CYP1A2 and CYP3A4 for 2-hydroxylation (20- and 18-fold higher,
respectively; Refs. 32
, 33
), which indicates that the E2
4-hydroxylation activity of CYP1B1 has the highest catalytic efficiency
of all of the reported E2 hydroxylases. Thus, CYP1B1 seems to be the
main cytochrome P450 responsible for the 4-hydroxylation of E2.
Previous studies of CYP1B1 have noted trace 16-hydroxylation
(29)
. Quantitation of the 16-hydroxylation activity of
wild-type CYP1B1 revealed a 15-fold lower level than the
4-hydroxylation activity (Table 2)
.
The variant enzymes exceeded wild-type CYP1B1 with respect to 2-,
4-, and 16-hydroxylation activity. The largest difference was
obtained for variant 2, which displayed 2.3- and 3.4-fold higher 2- and
4-hydroxylation activities, respectively, than the wild-type enzyme.
The 4-OH-E2:2-OH-E2 rate ratios of the variant enzymes ranged from 3.0
to 3.8 compared with 2.0 for wild-type CYP1B1. Thus, the variant forms
of CYP1B1 may contribute to higher tissue levels of 4-OH-E2.
Corroboration of our findings is provided by a recent publication by
Shimada et al. (34)
, who examined the
functional effect of two of the four CYP1B1 polymorphisms (codons 119
and 432) on estrogen metabolism. Shimada et al. expressed
the recombinant proteins in a bicistronic system linking the CYP1B1 and
NADPH-P450 reductase cDNAs and used bacterial membranes rather than
purified proteins for analysis. The
kcat and
Km values were at least 3- to 4-fold
lower than those presented in this study. The low
kcat values may be attributable to
poor coupling of CYP1B1 to NADPH-P450 reductase resulting either from
the presence of bacterially derived membrane lipids or insufficient
saturation with expressed reductase. The underestimation of the
Km values is apparent from the data
presented in Shimada et al. The reported
Km values ranged between 2.5 and 5.3
µM, whereas the lowest concentration used in
calculating these values was 20 µM. The high
sensitivity of our gas chromatography/mass spectrometry detection
methods allowed the acquisition of data points surrounding the
Km values and, therefore, should be a
more accurate representation of such catalytic parameters.
Nevertheless, qualitatively, both of the studies are in agreement that
the variants differ in activity and that Arg48, Ser119, Val432, Asn453
(our variant 2) seems to have the highest catalytic efficiency
for the 4-hydroxylation of estradiol.
Tissue-specific hydroxylation of E2 at C-4 may be especially important for the breast because of the potential carcinogenicity and the estrogenic activity of 4-OH-E2. Both 2-OH-E2 and 4-OH-E2 bind to estrogen receptor with affinities similar to E2 (35 , 36) . Treatment of MCF-7 breast cancer cells with 2-OH-E2 and 4-OH-E2 increased the rate of cell proliferation and the expression of estrogen-inducible genes such as progesterone receptor and pS2. Relative to E2 (100%), the effects of 2-OH-E2 and 4-OH-E2 on proliferation rate, progesterone receptor, and pS2 expression were 36 and 76%, 10 and 28%, 48 and 79%, respectively (36 , 37) . The lower estrogenic potency of 2-OH-E2 compared with 4-OH-E2 may be attributable to more rapid dissociation of the former from the estrogen receptor (38) .
Several studies examined E2 hydroxylation in breast tissue.
Osborne et al. (7)
demonstrated elevated
16-hydroxylation of E2 in mammary explants (terminal duct lobular
units) of four breast cancer patients. The increase in
16-hydroxylation activity was 4- to 5-fold higher compared with
normal terminal duct lobular units obtained from patients undergoing
reduction mammoplasty. No difference in enzyme activity was observed in
explants of mammary fat from cancer and control patients. Imoto
et al. (39)
examined 31 paired samples of
breast cancer and adjacent noncancerous tissue for E2 2- and
16-hydroxylation activities. 16-hydroxylation activity was
detected only in one-third of the tumors, and the activity in these was
not significantly different from that in the adjacent normal tissues.
In contrast, C-2 hydroxylation activity was present in most cancers but
significantly lower than in the adjacent tissues. Another study showed
that microsomes obtained either from normal breast tissue adjacent to
breast cancer or from reduction mammoplasty expressed comparable 2- and
4-hydroxylation activities with 4-OH-E2:2-OH-E2 ratios of 1.3 and 0.7,
respectively. In contrast, microsomes prepared from carcinoma and
fibroadenoma predominantly catalyzed hydroxylation of E2 at C-4 with
4-OH-E2:2-OH-E2 ratios of 3.8 and 3.7, respectively (20)
.
These results suggest differential regulation of the C-2, C-4, and
C-16 pathways in benign and malignant breast tissue. However, these
tissue studies were performed without knowledge of individual
CYP1B1 genotypes.
In the breast, CYP1A1 and CYP1B1 are responsible for the hydroxylation of estrogens to the 2-OH and 4-OH catechol estrogens. Several Phase II enzymes either inactivate catechol estrogens or protect against estrogen carcinogenesis by detoxifying products of oxidative damage that may arise on redox cycling of catechol estrogens (40) . The ubiquitous catechol-O-methyltransferase (COMT) inactivates 2-OH and 4-OH catechol estrogens by O-methylation (41) . Glutathione S-transferases (GSTs) inactivate catechol estrogen quinones by conjugation with glutathione (42) . Genetic variants of each of these enzymes involved in catechol estrogen metabolism have been identified, some with proven or suspected change in function. Inherited alterations in the activity of any of these enzymes hold the potential to define differences in catechol estrogen metabolism and, thereby, explain differences in breast cancer risk associated with estrogen carcinogenesis. However, it is evident that no single genotype can be linked to all breast cancers. This is not surprising given the fact that the enzymes involved in catechol estrogen metabolism also participate in environmental carcinogen metabolism (43) .
In conclusion, wild-type and variant CYP1B1 show significant
differences in estrogen hydroxylation activities, which may result in
different concentrations of 2-OH-E2, 4-OH-E2, and 16-OH-E2. Because
the CYP1B1 polymorphisms are inherited, they will dictate exposure
levels to these E2 metabolites for life. Given the carcinogenic and
estrogenic potential of 4-OH-E2 and possibly 16-OH-E2, one may
speculate that inheritance of certain CYP1B1 variants may contribute to
interindividual differences in breast cancer risk associated with
estrogen-mediated carcinogenesis.
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FOOTNOTES |
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1 Supported in part by NIH F32 CA79162
(I. H. H.), R35 CA44353 (F. P. G.), and P30 ES00267 (F. F. P.,
F. P. G.). 
2 To whom requests for reprints should be
addressed, at Department of Pathology, TVC 4918, Vanderbilt University
Medical Center, Nashville, TN 37232. Phone: (615) 343-9117; Fax: (615)
343-9563; E-mail: fritz.parl{at}mcmail.vanderbilt.edu
3 The abbreviations used are: E2,
17ß-estradiol; CYP1B1, cytochrome P450 1B1; CYP1A1, cytochrome P450
1A1; TMS, trimethylsilyl; Ni-NTA, nickel-nitrilotriacetic acid.
Received 12/14/99. Accepted 4/26/00.
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-hydroxylation in human breast tissue: a potential biomarker of breast cancer risk. J. Natl. Cancer Inst., 85: 1917-1920, 1993.-hydroxylase activity: is this a genotoxic or nongenotoxic biomarker in human breast cancer risk?. J. Natl. Cancer Inst., 85: 1888-1891, 1993., ß-unsaturated aldehyde products of radical reactions and lipid peroxidation by human glutathione transferases. Proc. Natl. Acad. Sci. USA, 91: 1480-1484, 1994.This article has been cited by other articles:
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 L. FONTANA, L. DELORT, L. JOUMARD, N. RABIAU, R. BOSVIEL, S. SATIH, L. GUY, J.-P. BOITEUX, Y.-J. BIGNON, A. CHAMOUX, et al. Genetic Polymorphisms in CYP1A1, CYP1B1, COMT, GSTP1 and NAT2 Genes and Association with Bladder Cancer Risk in a French Cohort Anticancer Res, May 1, 2009; 29(5): 1631 - 1635. [Abstract] [Full Text] [PDF]
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M. L. Cote, W. Yoo, A. S. Wenzlaff, G. M. Prysak, S. K. Santer, G. B. Claeys, A. L. Van Dyke, S. J. Land, and A. G. Schwartz Tobacco and estrogen metabolic polymorphisms and risk of non-small cell lung cancer in women Carcinogenesis, April 1, 2009; 30(4): 626 - 635. [Abstract] [Full Text] [PDF]
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J. Beuten, J. A.L. Gelfond, J. J. Byrne, I. Balic, A. C. Crandall, T. L. Johnson-Pais, I. M. Thompson, D. K. Price, and R. J. Leach CYP1B1 variants are associated with prostate cancer in non-Hispanic and Hispanic Caucasians Carcinogenesis, September 1, 2008; 29(9): 1751 - 1757. [Abstract] [Full Text] [PDF]
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 B. Diergaarde, J. D. Potter, E. R. Jupe, S. Manjeshwar, C. D. Shimasaki, T. W. Pugh, D. C. DeFreese, B. A. Gramling, I. Evans, and E. White Polymorphisms in Genes Involved in Sex Hormone Metabolism, Estrogen Plus Progestin Hormone Therapy Use, and Risk of Postmenopausal Breast Cancer Cancer Epidemiol. Biomarkers Prev., July 1, 2008; 17(7): 1751 - 1759. [Abstract] [Full Text] [PDF]
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S. Zienolddiny, D. Campa, H. Lind, D. Ryberg, V. Skaug, L. B. Stangeland, F. Canzian, and A. Haugen A comprehensive analysis of phase I and phase II metabolism gene polymorphisms and risk of non-small cell lung cancer in smokers Carcinogenesis, June 1, 2008; 29(6): 1164 - 1169. [Abstract] [Full Text] [PDF]
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 T. M. Sissung, R. Danesi, D. K. Price, S. M. Steinberg, R. de Wit, M. Zahid, N. Gaikwad, E. Cavalieri, W. L. Dahut, D. L. Sackett, et al. Association of the CYP1B1*3 allele with survival in patients with prostate cancer receiving docetaxel Mol. Cancer Ther., January 1, 2008; 7(1): 19 - 26. [Abstract] [Full Text] [PDF]
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 P Vineis, F Veglia, S Garte, C Malaveille, G Matullo, A Dunning, M Peluso, L Airoldi, K Overvad, O Raaschou-Nielsen, et al. Genetic susceptibility according to three metabolic pathways in cancers of the lung and bladder and in myeloid leukemias in nonsmokers Ann. Onc., July 1, 2007; 18(7): 1230 - 1242. [Abstract] [Full Text] [PDF]
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S. K. Holt, M. A. Rossing, K. E. Malone, S. M. Schwartz, N. S. Weiss, and C. Chen Ovarian Cancer Risk and Polymorphisms Involved in Estrogen Catabolism Cancer Epidemiol. Biomarkers Prev., March 1, 2007; 16(3): 481 - 489. [Abstract] [Full Text] [PDF]
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 A. R. Belous, D. L. Hachey, S. Dawling, N. Roodi, and F. F. Parl Cytochrome P450 1B1-Mediated Estrogen Metabolism Results in Estrogen-Deoxyribonucleoside Adduct Formation Cancer Res., January 15, 2007; 67(2): 812 - 817. [Abstract] [Full Text] [PDF]
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 V. Paracchini, S. Raimondi, I. T. Gram, D. Kang, N. A. Kocabas, V. N. Kristensen, D. Li, F. F. Parl, T. Rylander-Rudqvist, P. Soucek, et al. Meta- and Pooled Analyses of the Cytochrome P-450 1B1 Val432Leu Polymorphism and Breast Cancer: A HuGE-GSEC Review Am. J. Epidemiol., January 15, 2007; 165(2): 115 - 125. [Abstract] [Full Text] [PDF]
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Y. Tanaka, H. Hirata, Z. Chen, N. Kikuno, K. Kawamoto, S. Majid, T. Tokizane, S. Urakami, H. Shiina, K. Nakajima, et al. Polymorphisms of Catechol-O-Methyltransferase in Men with Renal Cell Cancer Cancer Epidemiol. Biomarkers Prev., January 1, 2007; 16(1): 92 - 97. [Abstract] [Full Text] [PDF]
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S. I. Berndt, N. Chatterjee, W.-Y. Huang, S. J. Chanock, R. Welch, E. D. Crawford, and R. B. Hayes Variant in Sex Hormone-Binding Globulin Gene and the Risk of Prostate Cancer Cancer Epidemiol. Biomarkers Prev., January 1, 2007; 16(1): 165 - 168. [Abstract] [Full Text] [PDF]
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M. H. Tao, Q. Cai, W. H. Xu, N. Kataoka, W. Wen, W. Zheng, Y. B. Xiang, Z.-F. Zhang, and X. O. Shu Cytochrome P450 1B1 and Catechol-O-Methyltransferase Genetic Polymorphisms and Endometrial Cancer Risk in Chinese Women Cancer Epidemiol. Biomarkers Prev., December 1, 2006; 15(12): 2570 - 2573. [Full Text] [PDF]
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P. S. Crooke, M. D. Ritchie, D. L. Hachey, S. Dawling, N. Roodi, and F. F. Parl Estrogens, enzyme variants, and breast cancer: a risk model. Cancer Epidemiol. Biomarkers Prev., September 1, 2006; 15(9): 1620 - 1629. [Abstract] [Full Text] [PDF]
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 T. M. Sissung, D. K. Price, A. Sparreboom, and W. D. Figg Pharmacogenetics and Regulation of Human Cytochrome P450 1B1: Implications in Hormone-Mediated Tumor Metabolism and a Novel Target for Therapeutic Intervention Mol. Cancer Res., March 1, 2006; 4(3): 135 - 150. [Abstract] [Full Text] [PDF]
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J. Thibaudeau, J. Lepine, J. Tojcic, Y. Duguay, G. Pelletier, M. Plante, J. Brisson, B. Tetu, S. Jacob, L. Perusse, et al. Characterization of Common UGT1A8, UGT1A9, and UGT2B7 Variants with Different Capacities to Inactivate Mutagenic 4-Hydroxylated Metabolites of Estradiol and Estrone Cancer Res., January 1, 2006; 66(1): 125 - 133. [Abstract] [Full Text] [PDF]
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T. A. Sellers, J. M. Schildkraut, V. S. Pankratz, R. A. Vierkant, Z. S. Fredericksen, J. E. Olson, J. Cunningham, W. Taylor, M. Liebow, C. McPherson, et al. Estrogen Bioactivation, Genetic Polymorphisms, and Ovarian Cancer Cancer Epidemiol. Biomarkers Prev., November 1, 2005; 14(11): 2536 - 2543. [Abstract] [Full Text] [PDF]
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J. R. Starr, C. Chen, D. R. Doody, L. Hsu, S. Ricks, N. S. Weiss, and S. M. Schwartz Risk of Testicular Germ Cell Cancer in Relation to Variation in Maternal and Offspring Cytochrome P450 Genes Involved in Catechol Estrogen Metabolism Cancer Epidemiol. Biomarkers Prev., September 1, 2005; 14(9): 2183 - 2190. [Abstract] [Full Text] [PDF]
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 A. DeMichele, R. Aplenc, J. Botbyl, T. Colligan, L. Wray, M. Klein-Cabral, A. Foulkes, P. Gimotty, J. Glick, B. Weber, et al. Drug-Metabolizing Enzyme Polymorphisms Predict Clinical Outcome in a Node-Positive Breast Cancer Cohort J. Clin. Oncol., August 20, 2005; 23(24): 5552 - 5559. [Abstract] [Full Text] [PDF]
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G. Li, Z. Liu, E. M. Sturgis, R. M. Chamberlain, M. R. Spitz, and Q. Wei CYP2E1 G1532C, NQO1 Pro187Ser, and CYP1B1 Val432Leu Polymorphisms Are Not Associated with Risk of Squamous Cell Carcinoma of the Head and Neck Cancer Epidemiol. Biomarkers Prev., April 1, 2005; 14(4): 1034 - 1036. [Full Text] [PDF]
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P. Kisselev, W.-H. Schunck, I. Roots, and D. Schwarz Association of CYP1A1 Polymorphisms with Differential Metabolic Activation of 17{beta}-Estradiol and Estrone Cancer Res., April 1, 2005; 65(7): 2972 - 2978. [Abstract] [Full Text] [PDF]
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W. Wen, Q. Cai, X.-O. Shu, J.-R. Cheng, F. Parl, L. Pierce, Y.-T. Gao, and W. Zheng Cytochrome P450 1B1 and Catechol-O-Methyltransferase Genetic Polymorphisms and Breast Cancer Risk in Chinese Women: Results from the Shanghai Breast Cancer Study and a Meta-analysis Cancer Epidemiol. Biomarkers Prev., February 1, 2005; 14(2): 329 - 335. [Abstract] [Full Text] [PDF]
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J. A. Doherty, N. S. Weiss, R. J. Freeman, D. A. Dightman, P. J. Thornton, J. R. Houck, L. F. Voigt, M. A. Rossing, S. M. Schwartz, and C. Chen Genetic Factors in Catechol Estrogen Metabolism in Relation to the Risk of Endometrial Cancer Cancer Epidemiol. Biomarkers Prev., February 1, 2005; 14(2): 357 - 366. [Abstract] [Full Text] [PDF]
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 S. Bandiera, S. Weidlich, V. Harth, P. Broede, Y. Ko, and T. Friedberg Proteasomal Degradation of Human CYP1B1: Effect of the Asn453Ser Polymorphism on the Post-Translational Regulation of CYP1B1 Expression Mol. Pharmacol., February 1, 2005; 67(2): 435 - 443. [Abstract] [Full Text] [PDF]
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J. L. Port, K. Yamaguchi, B. Du, M. De Lorenzo, M. Chang, P. M. Heerdt, L. Kopelovich, C. B. Marcus, N. K. Altorki, K. Subbaramaiah, et al. Tobacco smoke induces CYP1B1 in the aerodigestive tract Carcinogenesis, November 1, 2004; 25(11): 2275 - 2281. [Abstract] [Full Text] [PDF]
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 Y. Saijo, F. Sata, H. Yamada, K. Suzuki, S. Sasaki, T. Kondo, Y.Y. Gong, E.H. Kato, S. Shimada, M. Morikawa, et al. Ah receptor, CYP1A1, CYP1A2 and CYP1B1 gene polymorphisms are not involved in the risk of recurrent pregnancy loss Mol. Hum. Reprod., October 1, 2004; 10(10): 729 - 733. [Abstract] [Full Text] [PDF]
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T. Rylander-Rudqvist, S. Wedren, G. Jonasdottir, S. Ahlberg, E. Weiderpass, I. Persson, and M. Ingelman-Sundberg Cytochrome P450 1B1 Gene Polymorphisms and Postmenopausal Endometrial Cancer Risk Cancer Epidemiol. Biomarkers Prev., September 1, 2004; 13(9): 1515 - 1520. [Abstract] [Full Text] [PDF]
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M. McGrath, S. E. Hankinson, L. Arbeitman, G. A. Colditz, D. J. Hunter, and I. De Vivo Cytochrome P450 1B1 and catechol-O-methyltransferase polymorphisms and endometrial cancer susceptibility Carcinogenesis, April 1, 2004; 25(4): 559 - 565. [Abstract] [Full Text] [PDF]
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 M. Sasaki, Y. Tanaka, S. T. Okino, M. Nomoto, S. Yonezawa, M. Nakagawa, S. Fujimoto, N. Sakuragi, and R. Dahiya Polymorphisms of the CYP1B1 Gene as Risk Factors for Human Renal Cell Cancer Clin. Cancer Res., March 15, 2004; 10(6): 2015 - 2019. [Abstract] [Full Text] [PDF]
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S. S. Tworoger, J. Chubak, E. J. Aiello, C. M. Ulrich, C. Atkinson, J. D. Potter, Y. Yasui, P. L. Stapleton, J. W. Lampe, F. M. Farin, et al. Association of CYP17, CYP19, CYP1B1, and COMT Polymorphisms with Serum and Urinary Sex Hormone Concentrations in Postmenopausal Women Cancer Epidemiol. Biomarkers Prev., January 1, 2004; 13(1): 94 - 101. [Abstract] [Full Text] [PDF]
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D. L. Hachey, S. Dawling, N. Roodi, and F. F. Parl Sequential Action of Phase I and II Enzymes Cytochrome P450 1B1 and Glutathione S-Transferase P1 in Mammary Estrogen Metabolism Cancer Res., December 1, 2003; 63(23): 8492 - 8499. [Abstract] [Full Text] [PDF]
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T. Rylander-Rudqvist, S. Wedren, F. Granath, K. Humphreys, S. Ahlberg, E. Weiderpass, M. Oscarson, M. Ingelman-Sundberg, and I. Persson Cytochrome P450 1B1 gene polymorphisms and postmenopausal breast cancer risk Carcinogenesis, September 1, 2003; 24(9): 1533 - 1539. [Abstract] [Full Text] [PDF]
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M. Sasaki, Y. Tanaka, M. Kaneuchi, N. Sakuragi, and R. Dahiya CYP1B1 Gene Polymorphisms Have Higher Risk for Endometrial Cancer, and Positive Correlations with Estrogen Receptor {alpha} and Estrogen Receptor {beta} Expressions Cancer Res., July 15, 2003; 63(14): 3913 - 3918. [Abstract] [Full Text] [PDF]
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J. S. Mammen, G. S. Pittman, Y. Li, F. Abou-Zahr, B. A. Bejjani, D. A. Bell, P. T. Strickland, and T. R. Sutter Single amino acid mutations, but not common polymorphisms, decrease the activity of CYP1B1 against (-)benzo[a]pyrene-7R-trans-7,8-dihydrodiol Carcinogenesis, July 1, 2003; 24(7): 1247 - 1255. [Abstract] [Full Text] [PDF]
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S. Dawling, N. Roodi, and F. F. Parl Methoxyestrogens Exert Feedback Inhibition on Cytochrome P450 1A1 and 1B1 Cancer Res., June 15, 2003; 63(12): 3127 - 3132. [Abstract] [Full Text] [PDF]
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M. Yang, J.-Y. Jang, S. Kim, S.-M. Lee, S.-S. Chang, H.-K. Cheong, E. Lee, D. Kang, H. Kim, T. Kawamoto, et al. Genetic effects on urinary 1-hydroxypyrene levels in a Korean population Carcinogenesis, June 1, 2003; 24(6): 1085 - 1089. [Abstract] [Full Text] [PDF]
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L. C. Hodges, J. D. Cook, E. K. Lobenhofer, L. Li, L. Bennett, P. R. Bushel, C. M. Aldaz, C. A. Afshari, and C. L. Walker Tamoxifen Functions As a Molecular Agonist Inducing Cell Cycle-Associated Genes in Breast Cancer Cells Mol. Cancer Res., February 1, 2003; 1(4): 300 - 311. [Abstract] [Full Text] [PDF]
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 B. Bournique and A. Lemarie Docetaxel (Taxotere) Is Not Metabolized by Recombinant Human CYP1B1 in Vitro, but Acts as an Effector of This Isozyme. Drug Metab. Dispos., November 1, 2002; 30(11): 1149 - 1152. [Abstract] [Full Text] [PDF]
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I. De Vivo, S. E. Hankinson, L. Li, G. A. Colditz, and D. J. Hunter Association of CYP1B1 Polymorphisms and Breast Cancer Risk Cancer Epidemiol. Biomarkers Prev., May 1, 2002; 11(5): 489 - 492. [Abstract] [Full Text] [PDF]
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 M M de Jong, I M Nolte, G J te Meerman, W T A van der Graaf, J C Oosterwijk, J H Kleibeuker, M Schaapveld, and E G E de Vries Genes other than BRCA1 and BRCA2 involved in breast cancer susceptibility J. Med. Genet., April 1, 2002; 39(4): 225 - 242. [Abstract] [Full Text] [PDF]
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E. Aklillu, M. Oscarson, M. Hidestrand, B. Leidvik, C. Otter, and M. Ingelman-Sundberg Functional Analysis of Six Different Polymorphic CYP1B1 Enzyme Variants Found in an Ethiopian Population Mol. Pharmacol., March 1, 2002; 61(3): 586 - 594. [Abstract] [Full Text] [PDF]
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 S. A. Larsen-Su and D. E. Williams Transplacental Exposure to Indole-3-carbinol Induces Sex-Specific Expression of CYP1A1 and CYP1B1 in the Liver of Fischer 344 Neonatal Rats Toxicol. Sci., December 1, 2001; 64(2): 162 - 168. [Abstract] [Full Text] [PDF]
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Y.-J. Chun, S. Kim, D. Kim, S.-K. Lee, and F. P. Guengerich A New Selective and Potent Inhibitor of Human Cytochrome P450 1B1 and Its Application to Antimutagenesis Cancer Res., November 1, 2001; 61(22): 8164 - 8170. [Abstract] [Full Text] [PDF]
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S. Dawling, N. Roodi, R. L. Mernaugh, X. Wang, and F. F. Parl Catechol-O-Methyltransferase (COMT)-mediated Metabolism of Catechol Estrogens: Comparison of Wild-Type and Variant COMT Isoforms Cancer Res., September 1, 2001; 61(18): 6716 - 6722. [Abstract] [Full Text] [PDF]
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Y. Ko, J. Abel, V. Harth, P. Brode, C. Antony, S. Donat, H.-P. Fischer, M. E. Ortiz-Pallardo, R. Thier, A. Sachinidis, et al. Association of CYP1B1 Codon 432 Mutant Allele in Head and Neck Squamous Cell Cancer Is Reflected by Somatic Mutations of p53 in Tumor Tissue Cancer Res., June 1, 2001; 61(11): 4398 - 4404. [Abstract] [Full Text] [PDF]
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 G. Garcia-Cardena, J. Comander, K. R. Anderson, B. R. Blackman, and M. A. Gimbrone Jr. Inaugural Article: Biomechanical activation of vascular endothelium as a determinant of its functional phenotype PNAS, April 10, 2001; 98(8): 4478 - 4485. [Abstract] [Full Text] [PDF]
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) hydroxylation
Km 40 ± 8 µM,
kcat 4.4 ± 0.4
min-1; 2-OH-E2 (
) hydroxylation
Km 34 ± 4 µM,
kcat 1.9 ± 0.1
min-1; 16