0090-9556/02/3008-869–874$7.00
DRUG METABOLISM AND DISPOSITION
Copyright © 2002 by The American Society for Pharmacology and Experimental Therapeutics
DMD 30:869–874, 2002
Vol. 30, No. 8
661/995627
Printed in U.S.A.
METABOLISM OF TAMOXIFEN BY RECOMBINANT HUMAN CYTOCHROME P450
ENZYMES: FORMATION OF THE 4-HYDROXY, 4ⴕ-HYDROXY AND N-DESMETHYL
METABOLITES AND ISOMERIZATION OF trans-4-HYDROXYTAMOXIFEN
H. KIM CREWE, LISA M. NOTLEY, REBECCA M. WUNSCH, MARTIN S. LENNARD,
AND
ELIZABETH M. J. GILLAM
Section of Molecular Pharmacology and Pharmacogenetics, Division of Clinical Sciences (Central Sheffield University Hospital Trust), University
of Sheffield, Royal Hallamshire Hospital, Sheffield, United Kingdom (H.K.C., M.S.L.); and Department of Physiology and Pharmacology, School
of Biomedical Sciences, University of Queensland, St. Lucia, Australia (L.M.N., R.M.W., E.M.J.G.).
(Received December 17, 2001; accepted April 18, 2002)
This article is available online at http://dmd.aspetjournals.org
The cytochrome P450 (P450)-mediated biotransformation of tamoxifen is important in determining both the clearance of the drug
and its conversion to the active metabolite, trans-4-hydroxytamoxifen. Biotransformation by P450 forms expressed extrahepatically,
such as in the breast and endometrium, may be particularly important in determining tissue-specific effects of tamoxifen. Moreover,
tamoxifen may serve as a useful probe drug to examine the regioselectivity of different forms. Tamoxifen metabolism was investigated in vitro using recombinant human P450s. Forms CYP1A1,
1A2, 1B1, 2A6, 2B6, 2C9, 2C19, 2D6, 2E1, 3A4, 3A5, and 3A7 were
coexpressed in Escherichia coli with recombinant human NADPHcytochrome P450 reductase. Bacterial membranes were harvested
and incubated with tamoxifen or trans-4-hydroxytamoxifen under
conditions supporting P450-mediated catalysis. CYP2D6 was the
major catalyst of 4-hydroxylation at low tamoxifen concentrations
(170 ⴞ 20 pmol/40 min/0.2 nmol P450 using 18 ␮M tamoxifen), but
CYP2B6 showed significant activity at high substrate concentrations (28.1 ⴞ 0.8 and 3.1 ⴞ 0.5 nmol/120 min/0.2 nmol P450 for
CYP2D6 and CYP2B6, respectively, using 250 ␮M tamoxifen).
These two forms also catalyzed 4ⴕ-hydroxylation (13.0 ⴞ 1.9 and
1.4 ⴞ 0.1 nmol/120 min/0.2 nmol P450, respectively, for CYP2B6
and CYP2D6 at 250 ␮M tamoxifen; 0.51 ⴞ 0.08 pmol/40 min/0.2
nmol P450 for CYP2B6 at 18 ␮M tamoxifen). Tamoxifen N-demethylation was mediated by CYP2D6, 1A1, 1A2, and 3A4, at low substrate concentrations, with contributions by CYP1B1, 2C9, 2C19
and 3A5 at high concentrations. CYP1B1 was the principal catalyst
of 4-hydroxytamoxifen trans-cis isomerization but CYP2B6 and
CYP2C19 also contributed.
The nonsteroidal antiestrogen tamoxifen is currently the most
widely used chemotherapeutic agent for the adjuvant treatment of
breast cancer. It may act, at least in part, as a prodrug, by conversion
to the primary metabolite, trans-4-hydroxytamoxifen, which has enhanced binding affinity and potency compared with the parent compound (Katzenellenbogen et al., 1984). Tamoxifen has also been
shown to halve the risk of breast cancer in women with high susceptibility to the disease (Fisher et al., 1998). However, its prophylactic
benefit is compromised by a small but statistically significant increase
in the risk of developing endometrial cancer (International Agency for
Research on Cancer, 1996).
The P4501-mediated biotransformation of tamoxifen is important in
determining both the clearance of the drug and its conversion to the
active metabolite, trans-4-hydroxytamoxifen. Flavin-containing
monooxygenases appear to be responsible for N-oxide formation.
Tamoxifen has been shown to be metabolized by human P450s to
N-desmethyl, 4-hydroxy, and ␣-hydroxy metabolites. Previous studies
have pointed to CYP3A4 being the major catalyst of N-demethylation
(Jacolot et al., 1991; Crewe et al., 1997) with potential contributions
by CYP1A1 and 1A2 (Simon et al., 1993). Studies in one of our
laboratories (Crewe et al., 1997) and others (Dehal and Kupfer, 1997)
have suggested that CYP2D6 plays a predominant role in the 4-hydroxylation of tamoxifen, with contributions from CYP2C9 and 3A4.
In addition, secondary metabolites resulting from further metabolism of these derivatives may be formed by phase I processes. P450catalyzed isomerization of trans-4-hydroxytamoxifen has been reported (Williams et al., 1994), resulting in the conversion of a potent
antiestrogen (trans-4-hydroxytamoxifen) into a weak estrogen agonist
(cis-4-hydroxytamoxifen). Clinical resistance to tamoxifen therapy
has been associated with decreased tumor concentrations of tamoxifen
and an increase in the cis/trans ratio of 4-hydroxytamoxifen (Osborne
et al., 1992). Biotransformation of tamoxifen by P450 forms expressed extrahepatically, such as in the breast and endometrium, may
be particularly important in determining tissue-specific effects of the
drug, with respect to both the removal of parent drug and the generation of therapeutic or toxic metabolites.
The objectives of the current study were to determine which human
P450 forms might participate in the extrahepatic metabolism of tamoxifen to its N-desmethyl, 4-hydroxy, and 4⬘-hydroxy metabolites
and in the isomerization of trans-4-hydroxytamoxifen (Fig. 1). In
Funding for this study was provided by the Kathleen Cuningham Foundation
for Breast Cancer Research and the Australian Cancer Fund.
1
Abbreviations used are: P450, cytochrome P450 (heme-thiolate protein
P450); HPLC, high-performance liquid chromatography; hNPR, human NADPHcytochrome P450 reductase.
Address correspondence to: Dr. E. M. J. Gillam, Department of Physiology
and Pharmacology, School of Biomedical Sciences, The University of Queensland, St. Lucia, Queensland, Australia 4072. E-mail: [email protected]
869
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ABSTRACT:
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CREWE ET AL.
addition, since it is metabolized via several alternative pathways,
tamoxifen may serve as a useful probe drug for examining the regioselectivity of different P450 enzymes. Accordingly, the ability of
closely related forms to metabolize tamoxifen via its primary routes
was studied.
Materials and Methods
Chemicals and Drugs. The metabolite standards for HPLC analyses were
kindly provided by Dr. I. N. H. White (Medical Research Council, Toxicology
Unit, University of Leicester, UK; racemic 4-hydroxytamoxifen, N-desmethyltamoxifen, N,N-didesmethyltamoxifen, tamoxifen N-oxide) and Dr. P. Jank
(Klinge Pharma GmbH, Munich, Germany; N-desmethyldroloxifene, trans-4hydroxytamoxifen, racemic 4-hydroxytamoxifen, 4⬘-hydroxytamoxifen,
N-desmethyltamoxifen, N,N-didesmethyltamoxifen and tamoxifen N-oxide).
N,N-Didesmethyltoremifene was the generous gift of Orion-Farmos Corporation (Turku, Finland). Tamoxifen citrate, racemic and trans-4-hyroxytamoxifen, and nafoxidine were purchased from the Sigma-Aldrich (St. Louis, MO).
All other chemicals were obtained from local suppliers at the highest quality
commercially available.
Human Liver Microsomes. Samples of human liver were obtained from
organ donors according to procedures approved by University of Queensland
ethics committees and frozen in liquid nitrogen for storage at ⫺70°C prior to
use. Microsomes were prepared according to the method of Guengerich
(1994), with the addition of a final wash in resuspension buffer (10 mM
Tris-acetate, 1 mM EDTA containing 20% glycerol) to remove residual drugs.
P450 concentrations were determined as previously described (Guengerich,
1994).
Recombinant P450 Preparations. Human P450 enzymes were expressed
in bacteria in bicistronic format with hNPR as described previously (Gillam et
al., 1997; Parikh et al., 1997; Cuttle et al., 2000). DH5␣ strain Escherichia coli
Downloaded from dmd.aspetjournals.org at ASPET Journals on June 18, 2017
FIG. 1. Metabolic pathways of tamoxifen examined.
were transformed with bicistronic expression constructs each of which contained cDNAs encoding hNPR and one of the following recombinant P450s:
CYP1A1, 1A2, 1B1 [four variants, designated RAVN, RALN, GSVN, GSLN
expressing the following amino acids at positions 48 (Arg or Gly), 119 (Ala or
Ser), and 432 (Val or Leu); all four variants contained Asn at position 453
(Shimada et al., 2000)], CYP2A6, 2B6, 2C9*1 (wild type), CYP2C19, 2D6
[full-length variant designated DB4 (Gillam et al., 1995), but with wild type
(Ala) at position 374], CYP2E1, 3A4, 3A5, and 3A7. Cells were also transformed with the monocistronic expression vector containing the cDNA for
hNPR alone and with the empty vector, pCW. Bacteria were cultured and
harvested as described previously (Gillam et al., 1993) except that the expression of P450 forms CYP1A1, 2A6, 2B6, 2D6, and 3A7 was augmented by
coexpression of bacterial chaperones. Briefly, E. coli strain DH5␣ was cotransformed with the relevant bicistronic expression vector and pGro7 (Nishihara et al., 1998) using the method of Inoue et al. (1990). Colonies harboring
both plasmids were selected by growth on LB agar containing 100 ␮g/ml
ampicillin and 20 ␮g/ml chloramphenicol. Isolated colonies were precultured
at 37°C overnight in LB media containing both antibiotics then used to
inoculate terrific broth containing 1 mM thiamine, trace elements, 100 ␮g/ml
ampicillin, and 20 ␮g/ml chloramphenicol. Cultures were incubated for 5 h at
25°C, 200 rpm shaking speed before initiation of induction by the addition of
arabinose (4 mg/ml), isopropyl ␤-D-thiogalactoside (1 mM), and ␦⫺aminolevulinic acid (0.5 mM). Flasks were then incubated for a further 43 h at 25°C,
160 to 180 rpm before harvest. Membranes were prepared and characterized
for P450 hemoprotein expression and hNPR activity. Bacterial membranes
were used directly in enzyme assays.
Enzyme Assays. Incubations contained 0.05 to 0.2 ␮M P450 from microsomes or bacterial membranes, substrate, an NADPH-generating system (consisting of 1 mM NADPH, 2.5 mM glucose 6-phosphate, and 0.5 U/ml glucose6-phosphate dehydrogenase) in either 80 mM potassium phosphate, pH 7.4,
with 0.46% (w/v) KCl (experiments with 18 ␮M tamoxifen) or 100 mM
potassium phosphate, pH 7.4, with 2 mM ascorbic acid (experiments using 250
␮M tamoxifen). Incubations using CYP3A and CYP2C forms were supplemented with membranes obtained from cells expressing hNPR alone. Control
incubations were included in each assay run and contained membranes from
cells expressing hNPR alone (“hNPR”) or from cells transformed with the
empty expression vector (“pCW”). All procedures were undertaken under
reduced light and using amber glass and plasticware to minimize photodegradation of tamoxifen.
Assay of Primary Metabolites. Tamoxifen was added to the incubation
mixture from a methanolic stock solution to give a substrate concentration of
250 ␮M or from a stock in acetone/ethanol (1:1 v/v) to give a concentration of
18 ␮M. Final concentrations of solvent were maintained below 1% v/v in all
cases. Reactions were initiated by the addition of the NADPH-generating
system and incubated at 37°C with gentle agitation for 40 min (18 ␮M
experiments) or 120 min (250 ␮M experiments) unless otherwise indicated.
For incubations carried out with 18 ␮M tamoxifen, incubations (200 ␮l)
were terminated by addition of 1.8 ml of methyl-t-butyl ether and 50 ␮l of
internal standard (100 ␮g/ml nafoxidine). The aqueous and organic phases
were mixed vigorously then the upper 1.5 ml were removed and evaporated to
dryness. Residues were reconstituted in 250 ␮l of a mixture of mobile phase
and water (2:1 v/v) prior to analysis by HPLC (isocratic conditions).
For incubations carried out with 250 ␮M tamoxifen, incubations (1 ml)
were terminated by addition of 2 ml of helium-purged ethyl acetate and 100 ␮l
of internal standard (15 ␮M N,N-didesmethyl toremifene HCl). The phases
were mixed vigorously, and the upper 1.4 ml were removed. A second
extraction was performed with a further 1.2 ml of ethyl acetate. The combined
extracts were evaporated to dryness under argon, and the residue was reconstituted in 100 ␮l of acetonitrile prior to analysis by HPLC (gradient conditions).
Isomerization of trans-4-Hydroxytamoxifen. Membranes containing recombinant enzymes were incubated using the method described above with
minor modifications. trans-4-Hydroxytamoxifen was added as substrate (from
a stock in 1.15% w/v KCl) to a final concentration of 0.52 ␮M. Incubations
(500 ␮l total volume) were allowed to proceed at 37°C for 40 min before being
terminated by addition of 1.8 ml of ethyl acetate and 50 ␮l of internal standard
(0.75 ␮g/ml N-desmethyldroloxifene). The phases were mixed vigorously,
then the upper 1.4 ml was removed and evaporated under air. Residues were
TAMOXIFEN METABOLISM BY CYTOCHROME P450
Results
Metabolism of Tamoxifen to Its Primary Metabolites. The metabolism of tamoxifen to 4-hydroxy and N-desmethyl metabolites was
examined using a range of recombinant P450 forms. At the lower
substrate concentration tested (18 ␮M), 4-hydroxylation was catalyzed predominantly by CYP2D6 (170 ⫾ 20 pmol/40 min/0.2 nmol
P450), with minor contributions by CYP2B6, 2C9, 3A4, 1A1, 1A2,
and 3A5 (Fig. 2A). The relative contribution of CYP2B6 was increased at the high substrate concentration (250 ␮M; 28.1 ⫾ 0.8 and
3.1 ⫾ 0.5 nmol/120 min/0.2 nmol P450 for CYP2D6 and CYP2B6,
respectively; Fig. 2B). N-Demethylation of tamoxifen (Table 1) was
catalyzed by CYP2D6, 1A1, 1A2, and 3A4 at the lower concentration,
whereas CYP2D6 was the major catalysts at the high concentration,
with smaller contributions by CYP1A1, 1A2, 1B1 (all variants), 2C9,
2C19, 3A4, and 3A5.
Incubations with recombinant CYP2B6 and 2D6 produced an additional HPLC peak not observed with any other forms, which had the
same retention time as 4⬘-hydroxytamoxifen. This peak was quanti-
FIG. 2. 4-Hydroxylation of tamoxifen by recombinant P450 forms and human
liver microsomes.
Bacterial membranes containing recombinant P450s (0.2 ␮M P450) coexpressed
with reductase or liver microsomes were incubated with tamoxifen in the presence
or absence of an NADPH-generating system, and metabolites were analyzed by
HPLC. In all cases, incubations containing CYP3A and CYP2C forms were supplemented with membranes obtained from cells expressing hNPR alone. Control
incubations were included in each assay run and contained membranes from cells
expressing hNPR alone (“hNPR”) and from cells transformed with the empty
expression vector (“pCW”). Data represent the mean ⫾ S.D. of three determinations. A, incubations were carried out with 18 ␮M tamoxifen for 40 min, and
metabolites were analyzed using an isocratic method for separation. Closed bars
represent complete incubations; open bars represent NADPH-deficient control incubations. Asterisks signify data significantly different from controls (Student’s t
test) at the following levels: 多, p ⬍ 0.05; 多多, p ⬍ 0.01; 多多多, p ⬍ 0.005. B,
incubations were carried out with 250 ␮M tamoxifen for 120 min, and metabolites
were analyzed using a gradient method for separation. Data are means ⫾ S.D. for
three independent determinations; no apparent metabolite formation was noted in
NADPH-deficient controls. Asterisks signify data significantly different to controls
(Student’s t test) at the following levels: 多, p ⬍ 0.01; 多多, p ⬍ 0.005; 多多多, p ⬍
0.001.
fied by comparison to a standard curve prepared with authentic
metabolite. CYP2B6 generated 0.51 ⫾ 0.08 pmol/40 min/0.2 nmol
P450 (mean ⫾ S.D.; n ⫽ 3) 4⬘-hydroxytamoxifen at a substrate
concentration of 18 ␮M. At 250 ␮M tamoxifen, CYP2B6 and 2D6
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reconstituted in a mixture of mobile phase and water (2:1 v/v, 300 ␮l) prior to
analysis by HPLC (isomerization conditions). Isomerization was also examined at a trans-4-hydroxytamoxifen concentration of 100 ␮M, using the
conditions described above for incubations with tamoxifen at 250 ␮M, and
isomers were analyzed by HPLC (gradient conditions).
HPLC Analysis of Metabolites. Three sets of HPLC conditions were used
according to the different incubations described above.
Isocratic Conditions. HPLC was performed according to a previously
described method (Crewe et al., 1997) and using a 5-␮m Spherisorb ODS-1
reverse phase column (250 ⫻ 4.6 mm; Waters Corp., Milford, MA) maintained
at 40°C. The mobile phase consisted of 10 mM KH2PO4, pH 3.7, buffer/
methanol/acetonitrile (2.5:3:4.5 v/v), and the flow rate was 1.3 ml/min. The
column eluant was subjected to UV irradiation, and metabolites were subsequently detected by fluorescence at excitation and emission wavelengths of
260 and 375 nm, respectively. The retention times of tamoxifen, N-desmethyltamoxifen, 4-hydroxytamoxifen, 4⬘-hydroxytamoxifen, tamoxifen N-oxide,
and nafoxidine were 27.5, 21.8, 14.6, 16.2, 24.4, and 31.5 min, respectively.
Gradient Conditions. HPLC was performed using a Shimadzu HPLC
system (Shimadzu Scientific Instruments Inc., Columbia, MD) fitted with an
auto-injector and a 3.9 ⫻ 150 mm Waters Symmetry C8 reverse phase column
(Waters Corp.). The mobile phase was 20 mM ammonium acetate/acetonitrile
run on a gradient derived from that described in Poon et al. (1995) and
modified by extension of the second gradient step to optimize peak separation.
Initial conditions were 95:5 20 mM ammonium acetate/acetonitrile. The following steps were programmed: 0 to 4 min linear gradient to 80:20 ammonium
acetate/acetonitrile; 4 to 24 min linear gradient to 60:40 ammonium acetate/
acetonitrile; 24 to 60 min linear gradient to 35:65 ammonium acetate/acetonitrile; 60 to 70 min constant at 35:65 ammonium acetate/acetonitrile; 70 to 80
min linear gradient to 95:5 ammonium acetate/acetonitrile; and a final reequilibration at 95:5 ammonium acetate/acetonitrile for 10 min. The flow rate
was 0.75 ml/min. Metabolites were detected by absorbance at 280 nm. Under
these conditions, the retention times of tamoxifen and its main metabolites
were as follows: trans-4-hydroxytamoxifen, 43.8 min; cis-4-hydroxytamoxifen, 44.9 min; 4⬘-hydroxytamoxifen, 46.7 min; N,N-didesmethyltamoxifen,
52.7 min; N-desmethyltamoxifen, 55.7 min; tamoxifen N-oxide, 59.5 min;
tamoxifen, 59.6 min. Quantification of metabolites was performed with reference to standard curves prepared using authentic metabolites after correction
for recovery of the internal standard (N,N-didesmethyl toremifene HCl).
Isomerization Conditions. HPLC was performed according to a previously
described method (Williams et al., 1994), using a Novapak (Waters Corp.) 4
␮M C8 column. The mobile phase was 10 mM KH2PO4, pH 3.7, buffer/
methanol/acetonitrile (3:3:4 v/v) and containing 0.02% (v/v) triethylamine.
The flow rate was set to 1.5 ml/min. The column eluant was subjected to UV
irradiation, and metabolites were detected by fluorescence using an excitation
wavelength of 260 nm and a 375 nm emission filter. The retention times of
trans-4-hydroxytamoxifen, cis-4-hydroxytamoxifen, and N-desmethyldroloxifene were 13, 16, and 11 min, respectively.
871
872
CREWE ET AL.
TABLE 1
N-Demethylation of tamoxifen by recombinant P450 forms and liver microsomes
N-Desmethytamoxifen Produced
Enzyme Preparation
nmol/120 min/0.2nmol P450b
276 ⫾ 11
356 ⫾ 55
⬍0.5c
⬍0.5
⬍0.5
⬍0.5
⬍0.5
⬍0.5
⬍0.5
⬍0.5
468 ⫾ 28
111 ⫾ 7
⬍0.5
109 ⫾ 24
22 ⫾ 10
N.D.
60 ⫾ 22
20.1 ⫾ 3.0
14.7 ⫾ 1.4
17.5 ⫾ 2.4
18.9 ⫾ 0.6
12.3 ⫾ 0.9
13.0 ⫾ 1.3
⬍1
⬍1
7.2 ⫾ 0.1
11.5 ⫾ 1.0
63.3 ⫾ 2.0
16.6 ⫾ 0.3
22.9 ⫾ 0.5
N.D.
N.D.
16.3 ⫾ 2.2
N.D.
N.D., not determined.
a
Incubations were carried out with 18 ␮M tamoxifen for 40 min using a P450 concentration
of 0.2 ␮M. Data are the means of three determinations, corrected for apparent N-desmethyl
formation in NADPH-deficient controls. Errors represent the sum of the standard errors for
complete and NADPH-deficient controls.
b
Incubations were carried out with 250 ␮M tamoxifen for 120 min. Data are the means of
three determinations, corrected for apparent N-desmethyl formation in time ⫽ 0 controls. Errors
represent the sum of the standard errors for complete and time ⫽ 0 controls.
c
Apparent metabolite formation was not significantly higher (p ⬎ 0.01; Student’s t test) to
that in P450-deficient (hNPR) controls.
generated 13.0 ⫾ 1.9 and 1.4 ⫾ 0.1 nmol/120 min/0.2 nmol P450,
respectively.
A small amount of N,N-didemethytamoxifen was also observed in
incubations of 250 ␮M tamoxifen with CYP1A2 and 2D6, two forms
that were seen to produce significant N-desmethyltamoxifen.
Isomerization of trans-4-Hydroxytamoxifen. The trans to cis
isomerization of 4-hydroxytamoxifen was catalyzed predominantly by
CYP1B1 and to a lesser extent CYP2B6 and 2C19 (Fig. 3). Isomerization was time- and NADPH-dependent as shown in previous work
(Williams et al., 1994). For certain forms (3A4, 1A1, 2D6), interpretation of the isomerization data were complicated by the finding that
substrate at the low concentrations employed appeared to be metabolized to secondary metabolites. Accordingly, isomerization of trans4-hydroxytamoxifen was also examined at high substrate concentrations (100 ␮M trans-4-hydroxytamoxifen). Under these conditions,
CYP1A1, 1B1 (all variants), 2B6, 2D6, 3A4, and 3A5 all catalyzed
isomerization of trans-4-hydroxytamoxifen (results not shown).
Discussion
Previous studies have identified some of the P450 forms involved
in the metabolism of tamoxifen. Studies in one of our laboratories
(Crewe et al., 1997) using human liver microsomes and recombinant
P450s expressed in lymphoblastoid cell membranes suggested that
CYP2D6 plays a predominant role in the 4-hydroxylation of tamoxifen, with contributions from CYP2C9 and 3A4. In a later study using
100 ␮M tamoxifen, Dehal and Kupfer (1997) demonstrated 4-hydroxylation activity only with CYP2D6, using a combination of
recombinant P450s expressed in E. coli and/or lymphoblastoid cell
membranes. The current study extends these findings by using a wide
range of forms and confirms the participation of CYP2C9 and 3A4 as
well as CYP2D6 in tamoxifen 4-hydroxylation at low substrate concentrations. Additionally however, we have demonstrated the role of
CYP1A1, CYP2B6, and CYP2C19 as minor contributors to 4-hydroxylation and of CYP2B6 as a significant catalyst at high substrate
concentration. The present findings indicate that CYP2C9 and
FIG. 3. Isomerization of trans-4-hydroxytamoxifen by recombinant human P450s
and liver microsomes.
Membranes containing recombinant enzymes (0.1 ␮M P450) and liver microsomes (0.56 ␮M P450) were incubated for 40 min with 0.52 ␮M trans-4-hydroxytamoxifen, and metabolites were analyzed by HPLC. Controls were stopped immediately after the reaction was initiated (t ⫽ 0) or lacked NADPH (⫺NADPH). Data
represent the mean of three independent determinations and are expressed as a
proportion of the amount of trans-4-hydroxytamoxifen in controls corrected for the
amount of cis-4-hydroxytamoxifen in controls. Asterisks signify data significantly
different to controls (Student’s t test) at the following levels: 多, p ⬍ 0.01; 多多, p ⬍
0.005; 多多多, p ⬍ 0.001.
CYP3A4 are minor catalysts of tamoxifen 4-hydroxylation in vitro at
low concentrations. However, their relative contributions in vivo may
be more significant, due to the higher expression of these forms
compared with CYP2D6. The lack of activity of other forms, such as
CYP2B6 and 2C19, as observed by Dehal and Kupfer (1997) may be
the result of inefficient coupling of the enzymes with NPR in the
recombinant systems or due to limited assay sensitivity.
Previous studies have pointed to CYP3A4 being the major catalyst
of N-demethylation (Jacolot et al., 1991; Crewe et al., 1997) with
potential contributions by CYP1A1 and 1A2 (Simon et al., 1993). The
present work suggests CYP2D6, 1A1, and 1A2 may be more effective
catalysts than CYP3A4 on an equivalent P450 basis and that several
other P450s (CYP1B1, 2C9, 2C19, and 3A5) may also show significant activity at high substrate concentrations. Dehal and Kupfer
(1997) have also provided evidence to support a role for a number of
P450s in N-demethylation. However, it is unclear how the levels of
activity found in the earlier study compared with controls carried out
in the absence of P450 or cofactors, since all forms examined showed
some apparent activity, including forms not seen to have activity here,
when compared with P450-deficient controls. Most commercial tamoxifen stocks show low level contamination with N-desmethyltamoxifen, so some N-demethylated metabolite is evident in all incubations. Alternatively, the differences in the enzyme system
(membrane environment, ratios of redox partners) may explain these
conflicting results.
Our findings suggest CYP1B1 may be principally responsible for
the isomerization of the antiestrogen trans-4-hydroxytamoxifen to the
weakly estrogenic cis isomer, a reaction that may be associated with
the drug-resistant phenotype in human breast tumors (Osborne et al.,
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1A1
1A2
1B1 (RAVN)
1B1 (RALN)
1B1 (GSVN)
1B1 (GSLN)
2A6
2B6
2C9
2C19
2D6
3A4
3A5
HL24
HL25
HL27
HL32
pmol/40 min/0.2 nmol P450a
873
TAMOXIFEN METABOLISM BY CYTOCHROME P450
mRNA and/or protein level in normal breast tissue and tumors (Huang
et al., 1996; Hellmold et al., 1998). In particular, CYP2D6, a polymorphic form shown to have a predominant role in both 4-hydroxylation and N-demethylation of tamoxifen, was found to be apparently
expressed constitutively in extensive metabolizers but may be subject
to tissue-specific regulation at the level of mRNA splicing (Huang et
al., 1996). It would be of key interest to determine whether expression
of any of these P450s was altered in resistance compared with sensitive tumors, especially that of CYP1B1 and 2B6 as key catalysts of
trans-4-hydroxytamoxifen isomerization.
Endometrial expression of P450s may influence some of the adverse effects of the drug in this tissue. The expression of CYP1B1
mRNA has been consistently detected in the human uterus (Liehr et
al., 1995; Shimada et al., 1996) and specifically in the endometrium
(Hakkola et al., 1997; Vadlamuri et al., 1998). However, Murray et al.
(1997) observed CYP1B1 protein expression only in uterine tumors
and not in normal tissue. In addition, there is controversy over the
detection of mRNAs for other P450 forms in the uterus. Schuetz et al.
(1993) reported the detection of CYP3A7 but not CYP3A4 or
CYP3A5, and Vadlamuri et al. (1998) and Shimada et al. (1996)
reported CYP1A1 expression. In contrast, Hukkanen et al. (1998)
failed to find either CYP1A1 or CYP3A7 but did detect CYP2B6, 2C,
2E1, 3A4, and 3A5. Further studies are required to confirm the
expression of these forms in breast and uterus, at both the mRNA and
protein levels, and to determine whether and to what extent individual
P450 forms may mediate tamoxifen metabolism in these tissues.
Investigations are currently underway to determine the extent to
which extrahepatic tissues can metabolize tamoxifen. Such information should help in minimizing individual patient risk of developing
resistance to tamoxifen and of developing endometrial cancer.
Acknowledgments. Grateful thanks are extended to Drs. I. N. H.
White and P. Jank and to the Orion Farmos Corporation for the gift of
chemicals used in this study.
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not been well characterized. One study has suggested it has a higher
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(Ruenitz et al., 1984).
The data presented here raise the possibility that extrahepatic forms
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