CYP3A4-mediated oxygenation versus dehydrogenation of raloxifene

Article abstract of DOI:10.1021/bi902213r

Raloxifene was approved in 2007 by the FDA for the chemoprevention of breast cancer in postmenopausal women at high risk for invasive breast cancer. Approval was based in part on the improved safety profile for raloxifene relative to the standard treatment of tamoxifen. However, recent studies have demonstrated the ability of raloxifene to form reactive intermediates and act as a mechanism-based inhibitor of cytochrome P450 3A4 (CYP3A4) by forming adducts with the apoprotein. However, previous studies could not differentiate between dehydrogenation to a diquinone methide and the more common oxygenation pathway to an arene oxide as the most likely intermediate to inactivate CYP3A4. In the current work, ¹⁸O-incorporation studies were utilized to carefully elucidate CYP3A4-mediated oxygenation versus dehydrogenation of raloxifene. These studies established that 3′-hydroxyraloxifene is produced exclusively via CYP3A4-mediated oxygenation and provide convincing evidence for the mechanism of CYP3A4-mediated dehydrogenation of raloxifene to a reactive diquinone methide, while excluding the alternative arene oxide pathway. Furthermore, it was demonstrated that 7-hydroxyraloxifene, which was previously believed to be a typical O₂-derived metabolite of CYP3A4, is in fact produced by a highly unusual hydrolysis pathway from a putative ester, formed by the conjugation of raloxifene diquinone methide with a carboxylic acid moiety of CYP3A4, or other proteins in the reconstituted system. These findings not only confirm CYP3A4-mediated dehydrogenation of raloxifene to a reactive diquinone methide but also suggest a novel route of raloxifene toxicity.

Full text of DOI:10.1021/bi902213r

4466 Biochemistry 2010, 49, 4466–4475
DOI: 10.1021/bi902213r
CYP3A4-Mediated Oxygenation versus Dehydrogenation of Raloxifene†
Chad D. Moore, Christopher A. Reilly, and Garold S. Yost*
Department of Pharmacology and Toxicology, University of Utah, Salt Lake City, Utah 84112
Received December 27, 2009; Revised Manuscript Received April 19, 2010
ABSTRACT: Raloxifene was approved in 2007 by the FDA for the chemoprevention of breast cancer in
postmenopausal women at high risk for invasive breast cancer. Approval was based in part on the improved
safety profile for raloxifene relative to the standard treatment of tamoxifen. However, recent studies have
demonstrated the ability of raloxifene to form reactive intermediates and act as a mechanism-based inhibitor
of cytochrome P450 3A4 (CYP3A4) by forming adducts with the apoprotein. However, previous studies could
not differentiate between dehydrogenation to a diquinone methide and the more common oxygenation pathway
to an arene oxide as the most likely intermediate to inactivate CYP3A4. In the current work, 18O-incorporation
studies were utilized to carefully elucidate CYP3A4-mediated oxygenation versus dehydrogenation of
raloxifene. These studies established that 30-hydroxyraloxifene is produced exclusively via CYP3A4-mediated
oxygenation and provide convincing evidence for the mechanism of CYP3A4-mediated dehydrogenation
of raloxifene to a reactive diquinone methide, while excluding the alternative arene oxide pathway.
Furthermore, it was demonstrated that 7-hydroxyraloxifene, which was previously believed to be a typical
O2-derived metabolite of CYP3A4, is in fact produced by a highly unusual hydrolysis pathway from a putative
ester, formed by the conjugation of raloxifene diquinone methide with a carboxylic acid moiety of CYP3A4, or
other proteins in the reconstituted system. These findings not only confirm CYP3A4-mediated dehydrogenation
of raloxifene to a reactive diquinone methide but also suggest a novel route of raloxifene toxicity.
Breast cancer is the second most common form of cancer in
women and second most common cause of cancer mortality in
the United States (1). Tamoxifen, the prototypical SERM,1 has
been the mainstay treatment for hormone-dependent breast
cancer (2, 3) and more recently used as a chemopreventive agent
in women at risk of developing breast cancer (4). Despite
the effectiveness of tamoxifen in the treatment of breast cancer,
its use has been linked to an increased risk of endometrial
cancer (5-8) through formation of DNA adducts (9-11). It
has been proposed that toxicity of tamoxifen is caused by the
dehydrogenation of 4-hydroxytamoxifen (the active metabolite
of tamoxifen) to reactive intermediates, such as a quinone
methide (12-14) which forms DNA and protein adducts.
As a result of tamoxifen’s potential side effects, several second
generation SERMs have been developed to reduce potential
toxicities. One such SERM, raloxifene, was originally used
clinically for the treatment and prevention of osteoporosis in
postmenopausal women (15, 16). Due to recent studies and the
clinical trial for chemoprevention of breast cancer (STAR trial:
Study of Tamoxifen and Raloxifene) that have shown raloxifene
to be as effective as tamoxifen in reducing breast cancer, with
a reduced risk of endometrial cancer and blood clots (17-19),
the FDA approved raloxifene for the chemoprevention of
breast cancer in 2007. However, as with tamoxifen, recent work
has shown that the metabolism of raloxifene via cytochrome
P450 3A4 (CYP3A4) can generate several reactive quinone
species (20-22). Furthermore, raloxifene has been shown to be
a mechanism-based inactivator of CYP3A4, forming adducts
with the apoprotein (21, 23-25). Although the inactivating
species has not been explicitly identified, it is theorized that
dehydrogenation of raloxifene to a diquinone methide is respon-
sible for the inactivation of CYP3A4 (20-22, 26). The efficient
excretion of raloxifene by presystemic intestinal glucuronidation
decreases the potential for abnormally high concentrations that
could be toxic (27). Thus, it appears that this SERM may be
substantially safer than tamoxifen or other first-generation
SERMs. In fact, even though raloxifene reactive intermediates
bind extensively to microsomal proteins, it has been characterized
as “a nonhepatotoxic drug” in a recent comparison of drugs that
bind extensively to microsomal proteins, to agents that do not
bind extensively (28). In addition, an analogue of the new SERM,
arzoxifene, with a fluorine substituted for the hydroxyl group at
the critical 40-position that must have a hydroxyl group to be
dehydrogenated to a diquinone methide, was not metabolized to
an electrophilic intermediate (29). To facilitate the development
of less toxic SERMs, it is critical to fully elucidate the mechanisms
of CYP3A4-mediated metabolism of raloxifene and identify the
inactivating specie(s).
Recent studies have identified several oxygenated raloxifene
metabolites and several GSH adducts (20, 21). Despite the high
quality of these reports, due to the complexity of raloxifene
metabolism, they were unable to fully characterize CYP3A4-
mediated oxygenation versus dehydrogenation of raloxifene.
Specifically, the oxygenated metabolites and GSH adducts could
have been produced from either the epoxide or diquinone methide
intermediates (21). In this study, we utilized 18O-incorpora-
tion studies to determine that 30-hydroxyraloxifene (30-OHRA)
was formed directly via P450-mediated oxygenation. In contrast,
†This work was supported by the National Institutes of Health,
National Institute of General Medical Sciences (Grant GM074249).
*Correspondence should be addressed to this author. E-mail:
gyost@pharm.utah.edu. Phone: (801) 581-7956. Fax: (801) 585-3945.
1Abbreviations: SERM, selective estrogen receptor modulator; GSH,
glutathione; LC/MS, liquid chromatography-mass spectrometry; ESI,
electrospray ionization; CID, collision-induced dissociation; DOPC,
1,2-dioleoyl-sn-glycero-3-phosphocholine; DLPC, 1,2-dilauryl-sn-gly-
cero-3-phophocholine; DLPS, 1,2-diacyl-sn-glycerophospho-L-serine
r
pubs.acs.org/Biochemistry
Published on Web 04/20/2010
2010 American Chemical Society

Article
Biochemistry, Vol. 49, No. 21, 2010 4467
it was determined that 7-hydroxyraloxifene (7-OHRA) was not
formed via CYP3A-mediated oxygenation. Rather, 7-OHRA
was formed exclusively through dehydrogenation of raloxifene
to a diquinone methide intermediate. In a novel mechanism,
this reactive diquinone methide conjugates with a carboxylic
acid moiety of CYP3A4 or another protein and is subseq-
uently released by acid-catalyzed hydrolysis of the ester to form
7-OHRA.
system contained 50 pmol of purified CYP3A4, 100 pmol of
recombinant POR, 100 pmol of cytochrome b5 (Invitrogen,
Carlsbad, CA), 0.04% (w/v) sodium cholate, and 20 μg of lipid
mix (equal weights of DOPC, DLPC, and DLPS). The mixture
was gently shaken at room temperature for 10 min. To the system
were added potassium phosphate buffer (50 mM, pH 7.4), GSH
(4 mM), MgCl2 (15 mM), and substrate (50 μM) in a final volume
of 500 μL. The mixture was preincubated at 37 °C for 5 min, and
the reaction was initiated by the addition of 2 mM NAPDH.
The reaction was allowed to proceed for 30 min at 37 °C and then
was terminated by one of two methods. Method 1: addition of
60 μL of trichloroacetic acid (TCA). The mixture was vortexed,
followed by centrifugation at 21000g for 15 min to remove the
protein. Raloxifene and metabolites were extracted using C-18
Sep-Pak cartridges (Waters, Taunton, MA). The resulting eluate
was concentrated to dryness by evaporation under nitrogen and
reconstituted in 10% acetonitrile (v/v) for analysis via LC/MS.
Method 2: addition of 500 μL of cold methanol. The mixture was
vortexed, followed by centrifugation at 21000g for 15 min to
remove the protein. The supernatant was concentrated to dryness
by evaporation with nitrogen for analysis via LC/MS.
To investigate the effects of GSH on raloxifene metabolism,
raloxifene was incubated with a reconstituted system as described
above, with or without GSH. The mixture was preincubated at
37 °C for 5 min, and the reaction was initiated by the addition of
2 mM NADPH. The reaction was allowed to proceed for 10 min
at 37 °C and then terminated by the addition of 100 μL of 60%
TCA (v/v), containing 11β-hydroxytestosterone as an internal
standard. The samples were then extracted using method 1. All
incubations were performed five times (i.e., five separate recon-
stituted systems).
MATERIAL AND METHODS
Materials. Raloxifene, NADPH, reduced GSH, silver oxide,
H218O, and propionic acid were purchased from Sigma-Aldrich
(St. Louis, MO). 18O2 was purchased from Cambridge Isotope
Laboratories, Inc. (Andover, MA). 7-OHRA and 30-OHRA syn-
thesized standards (20) were generous gifts from Dr. Judy L. Bolton
(University of Illinois, Chicago). The purity of the standards was
1
confirmed with H NMR. All other chemicals for synthesis or
analysis were of analytical grade or equivalent and obtained at the
highest grade commercially available.
Instrumentation. LC/MS was conducted using a Thermo
LCQ Advantage MAX mass spectrometer, coupled with an LC
system consisting of a Finnigan Surveyor LC pump and Surveyor
Autosampler (Thermo Fisher Scientific, Waltham, MA). Elec-
trospray ionization (ESI) with positive ionization was utilized.
The source temperature was set to 250 °C, ionization voltage to
5 kV, capillary voltage to 45 V, and sheath gas (N2) flow rate of
50 units. Parameters for MS/MS by CID with helium gas were
as follows: activation amplitude at 35.0%, activation Q at 0.250,
activation time at 30 ms, and isolation width of 2 amu. Chro-
matography was conducted using a Phenomenex Gemini 3 μm
C6-phenyl (150 ꢀ 2.00 mm) reverse-phase column (Phenomenex
Inc., Torrance, CA). The mobile phase consisted of solvent A,
acetonitrile, and solvent B, 10% methanol containing 0.4%
formic acid (v/v/v). For raloxifene analysis, the mobile phase
was linear from 5% to 20% solvent A over 40 min, increasing to
100% solvent A over 10 min, at a flow rate of 0.2 mL/min.
Identification of raloxifene metabolites was based on product-ion
spectra obtained from CID of the [M þ H]þ ions to their product
ions previously described by Yu et al. (20) and Chen et al. (21)
LCQ Zoom scans (minimum of 10 scans) of the [M þ H]þ or
[M þ 2H]2þ ion for each metabolite were utilized to quantitate
isotope peak distribution. For testosterone analysis, the mobile
phase was linear from 5% to 70% solvent A over 35 min, inc-
reasing to 95% solvent A over 5 min, with a flow rate of 0.2 mL/
min. Identification of testosterone metabolites was based on
detection of the hydroxylated metabolites at [M þ H]þ of m/z 305
and by comparison of retention times to standards. For raloxi-
fene/carboxylic acid conjugate analysis, the mobile phase was
linear from 5% to 40% solvent A over 20 min, increasing to 95%
solvent A over 10 min, at a flow rate of 0.2 mL/min.
18O-Incorporation Studies. To measure the incorporation
of oxygen from water, incubations of CYP3A4 and raloxifene
were conducted as described above, with the substitution of
250 μL of H218O (97%). Reactions were terminated by method 1.
To account for fractional incorporation and to determine maxi-
mal incorporation from H218O, prior to termination, a 25 μL
aliquot from each incubation was removed and added to 2 mg of
2-chloronicotinolyl chloride (Alfa Aesar, Ward Hill, MA) to
form the acid product. To this, anhydrous acetonitrile (100 μL)
was added, and the resulting mixture was analyzed by direct
infusion into the LCQ. The ratio of 16O to 18O incorporated into
the acid product was calculated using Brauman’s least-squares
method (32, 33). Briefly, the contribution of each isotope peak
for a compound was determined by primary Zoom scans of
unlabeled standard. The ion intensities of the 18O-labeled com-
pounds were corrected for the ion overlap due to normal isotopic
species, by subtracting the percent contribution of each isotope
from the compound of interest. This resulted in a series of simul-
taneous equations, which were solved by Brauman’s method
to calculate the percent of molecules containing 18O versus the
percent of molecules that contained 16O.
To measure the incorporation of oxygen from molecular
oxygen, incubations were conducted in an airtight four-flask
manifold apparatus that was constructed so that it could be
evacuated, flushed with ultrapure nitrogen gas, and repressurized
with 18O2 introduced. The incubations were conducted as de-
scribed above, with the following modifications. The potassium
phosphate buffer was purged with nitrogen prior to addition to
the reconstituted system. The manifold was then evacuated and
flushed with ultrapure nitrogen three times to remove any atmo-
spheric oxygen. 18O2 (97%) was then introduced to the system.
Preparation of Reconstituted CYP3A4 and Oxidoreduc-
tase. A CYP3A4 construct containing a C-terminal polyhistidine
tag cloned into the pSE380 vector was a generous gift from
Dr. James R. Halpert (University of California, San Diego, CA),
and the rat cytochrome P450 oxidoreductase (POR) construct
pOR262 was a generous gift from Dr. Charles B. Kasper
(University of Wisconsin, Madison, WI). CYP3A4 was expressed
in Escherichia coli DH5-R cells (Invitrogen, Carlsbad, CA) and
purified as described previously (30). Rat POR was expressed in
JM-109 cells and purified as described previously (31).
Incubation of Raloxifene, 7-Hydroxyraloxifene, and 30-Hy-
droxyraloxifene with Recombinant CYP3A4. The reconstituted

4468 Biochemistry, Vol. 49, No. 21, 2010
Moore et al.
The incubations (one per flask) were preincubated at 37 °C for
5 min, and the reactions were initiated by the addition of 2 mM
NAPDH via syringe through airtight septa. Reactions were
terminated by method 1. To account for fractional incorpora-
tion and determine maximal incorporation from 18O2, 200 μM
testosterone replaced raloxifene in one of the incubations. The
testosterone incubation was analyzed via LC/MS, and the ratio
of 16O to 18O incorporated into 6β-hydroxytestosterone was
calculated using Brauman’s least-squares method.
Chemical Oxidation Studies. To chemically form GS-
raloxifene, the procedure from Yu et al. (20) was utilized. Briefly,
raloxifene (5 mg) was dissolved in 5 mL of anhydrous acetonitrile
and heated to 60 °C. Silver oxide (0.5 g) was added and stirred for
10 s. The resulting slurry was immediately filtered into a secondary
solution consisting of 10 mL of 50 mM potassium phosphate
buffer (pH 7.4) containing 0.5 mM GSH. The secondary solution
was divided into one portion, which was concentrated under
nitrogen gas for analysis via LC/MS, and a second portion to
which TCA was added to a final concentration of 12% (v/v) and
concentrated under nitrogen gas for analysis via LC/MS.
To form the raloxifene/carboxylic acid conjugate, raloxifene
(5 mg) and 1 μL of glacial acetic acid or propionic acid was added
to 4 mL of anhydrous acetonitrile and heated to 60 °C. Silver
oxide (0.5 g) was subsequently added and stirred for 10 s. The
resulting slurry was immediately filtered into a secondary solu-
tion consisting of 5 mL of 50 mM potassium phosphate buffer
(pH 8.0). The secondary solution was divided in half, one portion
was concentrated under nitrogen gas for analysis via LC/MS, and
TCA was added to a second portion to produce a final concen-
tration of 12% (v/v) and concentrated under nitrogen gas for
analysis via LC/MS.
F
IGURE 1: Positive ESI LC/MS mass chromatograms of raloxifene
metabolites and GSH conjugates from the incubation of raloxifene
with CYP3A4. Di-GS-OHRA [Mþ2H]2þ ions were monitored at
m/z 550.5, GS-OHRA [M þ H]þ ionsweremonitored atm/z 795, GS-
RA [M þ H]þ ions were monitored atm/z 779, OHRA [M þ H]þ ions
were monitored at m/z 490, and raloxifene [Mþ H]þ ions were
monitored at m/z 474.
at m/z 795 produced fragment ions at m/z 522, 666, and 777,
corresponding to cleavage of the thioester, loss of pyroglu-
tamate, and loss of water, respectively (Figure 2B). There were
no differences between the MS/MS spectra of GS-OHRA-1 and
GS-OHRA-2. One mono-GS-raloxifene (GS-RA) metabolite
was identified based on the detection of a strong [M þ H]þ peak
at m/z 779 at 30.7 min (Figure 1). MS/MS analysis of the molecule
at m/z 779 produced strong fragment ions at m/z 650 and 761,
corresponding to losses of pyroglutamate and water, respectively
(Figure 2C). Weak fragment ions at m/z 506 and 686 corre-
sponded to a cleavage adjacent to the thioester moiety leaving
sulfur on the raloxifene and to the loss of glycine with water.
Two hydroxyraloxifene metabolites were identified based on the
detection of two [M þ H]þ peaks at m/z 490 at 32.9 and 34.6 min
(Figure 1). MS/MS analysis of both molecules at m/z 490
produced only one strong fragment ion at m/z 285, corresponding
to the loss of 1-(2-phenoxyethyl)piperidine (Figure 2D). Addi-
tional MSn analysis of these metabolites was unable to produce
fragment ions that could differentiate 7-OHRA and 30-OHRA.
However, the assignment of 7-OHRA to the peak at 32.9 min and
3-OHRA to the peak at 34.6 min was confirmed by comparison of
retention times to synthetic standards.
18O-Labeled propionic acid was synthesized by incubating
5 μL of [16O]propionic acid in 100 μL of H218O (97%, pH 1 with
HCl). The oxygen exchange proceeded for 3 days at 65 °C. The
solution was concentrated to dryness under nitrogen and recon-
stituted in 10 μL of anhydrous acetonitrile. Incorporation of 18O
was 90%, measured by direct infusion into the MS. The chemical
oxidation of raloxifene was repeated, with the exception that 5 μL
of the 18O-labeled propionic acid solution was added to the
anhydrous acetonitrile solution. The products were analyzed via
LC/MS.
RESULTS
Incubation of Raloxifene and Hydroxyraloxifenes with
Recombinant CYP3A4. Incubations of raloxifene with recom-
binant CYP3A4, terminated by method 1, were analyzed by
LC/MS/MS to identify raloxifene metabolites (Figure 1). We
identified a di-GS-hydroxyraloxifene (di-GS-OHRA) metabo-
lite, based on the detection of a strong [M þ 2H]2þ peak at m/z
550.5 at 18.3 min (a much weaker [M þ H]þ peak at m/z 1101 was
detected also at 18.3 min). MS/MS analysis of the diprotonated
molecule at m/z 550.5 produced doubly charged fragment ions at
m/z 486 and 421.5, corresponding to losses of one and two
pyroglutamate moieties from GSH, respectively (Figure 2A). The
doubly charged fragment ions at m/z 513 and 448.5 corresponded
to a loss of one glycine and to the concurrent loss of glycine and
pyroglutamate. The doubly charged fragment ions at m/z 541.5
and 477 corresponded to a loss of water and the concurrent loss
of water and pyroglutamate. Two GS-hydroxyraloxifene meta-
bolites (assigned GS-OHRA-1 and GS-OHRA-2) were identified
based on the detection of two weak [M þ H]þ peaks at m/z 795 at
22.8 and 25.3 min (Figure 1). MS/MS analysis of the molecules
Incubations of raloxifene with CYP3A4, terminated by meth-
od 2, were analyzed via LC/MS/MS to identify raloxifene meta-
bolites. Similar to the incubation terminated with acid, we were
able to detect a single di-GS-OHRA, two GS-OHRA, and a
single GS-RA metabolite. Retention times and MS/MS analysis
of the metabolites were the same as described previously.
However, in contrast to the acid-terminated incubations, only
30-OHRA was detected in incubations terminated by methanol
(Figure 3). Interestingly, when the protein pellets from the
methanol-terminated incubations were resuspended in 50 mM
potassium phosphate buffer (pH 7.4) and treated with acid, we
were able to detect appreciable amounts of previously undetect-
able 7-OHRA. Lesser amounts of raloxifene and raloxifene
metabolites, including 30-OHRA, were also detected, but these
molecules are believed to be adventitiously bound to the protein/
lipid pellet and released during secondary acid treatment.
7-OHRA was the only raloxifene metabolite which required acid
treatment to be liberated and detected.

Article
Biochemistry, Vol. 49, No. 21, 2010 4469
F
IGURE 2: Positive ESI product-ion mass spectra from CID of the raloxifene metabolites and GSH conjugates shown in Figure 1. Spectra were
obtained from CID of (A) di-GS-OHRA atm/z 550.5, (B) GS-OHRA atm/z 795, (C) GS-RA at m/z 779, and (D) OHRA at m/z 490. Mass spectra
for GS-OHRA-1 and -2 were identical; therefore, only a representative spectrum of GS-OHRA-1 is shown. Mass spectra for 7-OHRA and
30-OHRA were identical; therefore, only a representative spectrum of 7-OHRA is shown.
F
IGURE 3: Positive ESI LC/MS mass chromatograms of hydroxy-
raloxifene metabolites (m/z 490) from incubations of raloxifene with
CYP3A4. The top chromatogram shows that both 7-OHRA and
30-OHRA are detected after the incubation was terminated with
TCA. The middle chromatogram shows that only 30-OHRA was
detected after the incubation was terminated with methanol. In the
bottom chromatogram, the protein pellet from the methanol termi-
nation was resuspended in a TCA solution, and both 30-OHRA and
7-OHRA were recovered from the protein pellet.
F
IGURE 4: The effects of glutathione on CYP3A4-mediated produc-
tion of 7-hydroxyraloxifene and 30-hydroxyraloxifene with incuba-
tion terminated by addition of TCA. The relative amounts of
metabolites were estimated with an internal standard, and concen-
tration is expressed in arbitrary units (AU). (*) and (#) indicate
significant difference ( p < 0.05, n = 5).
To investigate the role of GSH in the metabolism of raloxifene,
incubations were conducted in the presence and absence of GSH.
These incubations were terminated by method 1, and the relative
amounts of hydroxylated metabolites were quantified by use of an
internal standard. The presence of GSH had no significant effect on
the production of 30-OHRA (Figure 4). However, the production
of 7-OHRA was significantly decreased (55%) in the presence of
GSH, compared to production of 7-OHRA in the absence of GSH.
Incubations of 7-OHRA and 30-OHRA standards with re-
combinant CYP3A4, terminated by method 1, were analyzed by
LC/MS and LC/MS/MS to identify metabolites (Figure 5).
Similar to previous studies with rat and human liver microsomes
by Yu et al. (20), the metabolism of 7-OHRA produced one
mono-GSH conjugate (GS-7-OHRA), identified by detection of
the [M þ H]þ peak at m/z 795 at 25.3 min and one di-GSH
conjugate (di-GS-7-OHRA) identified by detection of the [M þ
2H]2þ peak with m/z 550.5 at 18.5 min (Figure 5A). CYP3A4-
mediated metabolism of 30-OHRA produced two mono-GSH
conjugates (GS-30-OHRA-1 and GS-30-OHRA-2), identified
by detection of two [M þ H]þ peaks at m/z 795 at 23.3 and

4470 Biochemistry, Vol. 49, No. 21, 2010
Moore et al.
25.6 min, and one di-GSH conjugate (di-GS-30-OHRA) identi-
fied by detection of a [M þ 2H]2þ peak at m/z 550.5 at 18.7 min
(Figure 5B). MS/MS analysis of the GSH conjugates produced
spectra similar to that of the GSH conjugates produced from
CYP3A4-mediated metabolism of raloxifene (data not shown).
Based on identical retention times, GS-OHRA-1 eluting at
22.8 min in Figure 1 was determined to be hydroxylated at the
30-position. However, the hydroxylation site of GS-OHRA-2,
eluting at 25.3 min in Figure 1, could not be assigned unequi-
vocally. Similarly, the hydroxylation site of the di-GS-OHRA
metabolite in Figure 1 could not be assigned.
bated with CYP3A4 in the presence of 18O2. The incubations
were terminated by method 1 and analyzed via LC/MS utilizing
Zoom scans to quantitate isotopic abundance. Maximal possible
18O incorporation in the raloxifene metabolites was determined
to be 45%, by measuring the 18O incorporated into the positive
control, 6β-hydroxytestosterone. By inspection of the 30-OHRA
mass spectrum, it was apparent from the increase of the þ2
isotope peak (m/z 492) that 30-OHRA efficiently incorporated
18O (Figure 6A). Using Brauman’s least-squares method and
accounting for fractional incorporation, greater than 99% of
30-OHRA incorporated oxygen from O2 (i.e., P450-mediated
oxygenation). By inspection of the 7-OHRA and GS-RA mass
spectra, the absence of an increase in the þ2 isotope peaks (m/z
492 and 781, respectively) indicates that 7-OHRA and GS-RA
did not incorporate 18O (Figure 6B,C). Using Brauman’s least-
squares method no appreciable amount of oxygen in 7-OHRA or
GS-RA originated from O2. It is apparent from the increase of
the þ2 isotope peak (m/z 551.5, due to diprotonation) that di-GS-
OHRA incorporated 18O (Figure 6D). Using Brauman’s least-
squares method and accounting for fractional incorporation,
greater than 99% of di-GS-OHRA incorporated oxygen from
O2. Due to the minimal amounts of GS-OHRA metabolites
formed, we were unable to measure the amount of 18O incorpora-
tion by Brauman’s least-squares method. However, by visual
inspection, the GS-OHRA metabolites appeared to incorporate
an appreciable amount of 18O.
18O-Incorporation Studies. To determine the source of
oxygen for the hydroxylated metabolites, raloxifene was incu-
To determine if any of the hydroxylated raloxifene meta-
bolites were produced from hydration of a reactive intermediate,
raloxifene was incubated with CYP3A4 in the presence of H218O.
The incubations were terminated by method 1 and analyzed via
LC/MS with Zoom scan to quantitate the isotope peak distribu-
tion. Maximal possible 18O incorporation into the raloxifene
metabolites was determined to be 50%, by measuring the 18O
F
IGURE 5: Positive ESI LC/MS mass chromatograms of the GSH
conjugates from incubation of CYP3A4 with (A) 7-OHRA standard
and (B) 30-OHRA standard. All incubations were terminated by
addition of TCA. Di-GS-OHRA [M þ 2H]2þ ions were monitored at
m/z 550.5, and GS-OHRA [M þ H]þ ions were monitored atm/z 795.
F
IGURE 6: PositiveESI Zoom scan massspectra ofraloxifenemetabolites and GSH conjugates fromthe incubationof raloxifenewith CYP3A4in
the presence of 18O2 or H218O. Maximal possible 18O incorporation in the raloxifene metabolites was determined to be 45% from 18O2 and 50%
from H218O by measuring the 18O incorporated into the controls, 6β-hydroxytestosterone and 2-chloronicotinolyl acid, respectively. The isotope
patterns are shown for (A) 30-OHRA, (B) 7-OHRA, (C) GS-RA, and (D) di-GS-OHRA.

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Biochemistry, Vol. 49, No. 21, 2010 4471
F
IGURE 7: Positive ESI product-ion mass spectra from CID of the (A) raloxifene/acetic acid conjugate, [M þ H]þ = m/z 532, and (B) raloxifene/
propionic acid conjugate, [M þ H]þ = m/z 546, produced by adding chemically synthesized raloxifene diquinone methide in the presence of the
carboxylic acids. (*) indicates fragment ions that increased by 2 amu, and (þ) indicates fragment ions that increased by 4 amu when 18O-labeled
propionic acid was used to produce the adduct, [M þ H]þ = m/z 550.
incorporated into 2-chloronicotinolyl acid. By visual inspection,
due to the lack of an increase in their þ2 isotope peaks, it was
concluded that 30-OHRA, 7-OHRA, and GS-RA did not incor-
porate 18O from water (Figure 6A-C). Using Brauman’s least-
squares method and accounting for fractional incorporation, no
appreciable amount of oxygen was found to originate from H2O
in these metabolites. Careful inspection of the di-GS-OHRA
mass spectra showed a small, but appreciable, increase in the þ2
isotope peak (Figure 6D). Using Brauman’s least-squares meth-
od and accounting for fractional incorporation, it was deter-
mined that roughly 20% of di-GS-OHRA incorporated oxygen
from H2O. Due to the minute amounts of GS-OHRA metabo-
lites formed, we were unable to accurately measure the amount of
18O incorporation from H218O. However, the GS-OHRA meta-
bolites did not appear to incorporate an appreciable amount of
18O from water.
Chemical Oxidation. Silver oxide was used to chemically
oxidize raloxifene to a diquinone methide. The raloxifene diqui-
none methide is very unstable, with a half-life estimated to be less
than 1 s in phosphate buffer (pH 7.4) at 5 °C (20). To assay for
this unstable electrophilic diquinone methide, it was added to a
secondary solution containing GSH to form stable GSH con-
jugates. The resulting mixture was separated into two portions;
one was immediately analyzed, while TCA was added to the
second portion prior to analysis by LC/MS/MS. The raloxifene
diquinone methide formed a single GSH adduct, which was
identified as GS-RA by comparison of its retention time and MS/
MS spectrum to the GS-RA metabolite produced from CYP3A4-
mediated metabolism of raloxifene (data not shown). No hydro-
xyraloxifene peaks were detected, and the addition of TCA had
no effect on the product profile, demonstrating that the di-
quinone methide did not conjugate with the carboxyl groups
on GSH.
corresponded to the loss of the 1-(2-phenoxyethyl)piperidine
moiety and concurrent cleavage of the ester, leaving an oxygen.
The fragment ions at m/z 405 and 387 corresponded to the loss of
piperidine and the cleavage of the ester, leaving oxygen, and
concurrent loss of water. The fragment ion at m/z 472 corre-
sponded to the loss of the acetic acid moiety. The fragment ion at
m/z 490 corresponded to the cleavage of the ester, leaving oxygen.
MS/MS analyses of the m/z 546 peak produced fragment ions
similar to that of the m/z 532 peak, including at m/z 285, 387, 405,
and 490, which corresponded to the same fragment pattern as
discussed above (Figure 7B). However, the m/z 546 peak also had
fragment ions at m/z 341 and 363. The fragment ion at m/z 341
corresponded to the loss of the 1-(2-phenoxyethyl)piperidine
moiety. The fragment ion at m/z 363 corresponded to the
concurrent loss of 1-ethylpiperidine and propionic acid. LC/
MS/MS analysis of the TCA-treated samples detected a single
hydroxyraloxifene peak, [M þ H]þ at m/z 490, with a fragment
ion at m/z 285. This hydroxyraloxifene product was identified as
7-OHRA by comparison to the synthetic standard.
The raloxifene diquinone methide was trapped by incubation
with 18O-labeled propionic acid (incorporation of two oxygen
atoms) and analyzed by LC/MS. A peak with the putative
molecular weight of raloxifene conjugated to 18O-labeled pro-
pionic acid, [M þ H]þ = m/z 550, eluted at the same retention
time as the adduct formed with unlabeled propionic acid (m/z
546, raloxifene conjugated to [16O]propionic acid; data not
shown). MS/MS analysis of the labeled adduct produced frag-
ment ions (identified in Figure 7B) that were highly similar to the
unlabeled adduct, but with mass shifts of 2 or 4 amu, consistent
with the addition of one or two 18O atoms. Major fragment ions
of the adduct peak with a mass of m/z 550 showed mass shifts of
m/z 285 f 287 (þ1 18O), m/z 405 f 407 (þ1 18O), m/z 490 f 492
(þ1 18O), and m/z 341 f 345 (þ2 18O). These results confirmed
the identity of the diquinone methide, produced by chemical
oxidation of raloxifene, and its ability to be trapped with
carboxylic acids to form ester adducts.
To determine if raloxifene diquinone methide could form
conjugates with compounds containing carboxyl groups, the
electrophilic diquinone methide was formed in the presence of
either acetic acid or propionic acid. The resulting mixtures were
separated into two portions, one was immediately analyzed and
the other acidified with TCA prior to analysis via LC/MS/MS.
Analysis of the non-TCA-treated samples resulted in the detec-
tion of an ion consistent with the putative molecular weight of
raloxifene conjugated to acetic acid, [M þ H] þ=m/z 532, and
propionic acid, [M þ H] þ=m/z 546. MS/MS analyses of the m/z
532 peak produced fragment ions at m/z 285, 327, 387, 405, 447,
472, and 490 (Figure 7A). The fragment ions at m/z 327 and 285
DISCUSSION
Raloxifene is a second generation SERM used for the treat-
ment of osteoporosis in postmenopausal women (15, 16) and for
the chemoprevention of breast cancer. However, P450-mediated
metabolism of raloxifene produces reactive o-quinones and
multiple GSH adducts (20, 21). Raloxifene is also a mechanism-
based inactivator of CYP3A4, forming at least two adducts

4472 Biochemistry, Vol. 49, No. 21, 2010
Moore et al.
Scheme 1: Formation of Raloxifene Adducts with GSHa
aThe monoglutathione-raloxifene adduct at the C7 carbon is shown to be produced through CYP3A4-mediated epoxidation at the
C6-C7 position, followed by gem-diol intermediate formation at the C6 position. *O indicates 18O from molecular oxygen, where 50%
of the monoglutathione adduct would theoretically incorporate 18O from molecular oxygen through formation of the C6-C7 epoxide.
Conversely, epoxidation at the C4-C5 position (not shown), followed by epoxide hydration and aromatization through water loss
would produce the C5-C6 catechol. Oxidation of the catechol would form the o-quinone at C5-C6, which could trap GSH to form an
adduct that would theoretically fail to incorporate 18O from molecular oxygen. However, the adduct would have an additional hydroxyl
group, most likely at C5, and the adduct that was detected did not.
with the apoprotein (21, 23-25). The current study also demon-
strates that CYP3A4 dehydrogenates raloxifene to form a
highly reactive diquinone methide. Due to the instability of this
electrophile, it can only be detected by trapping it as its mono-
GSH adduct (GS-RA). The origins of this adduct could arise
either from dehydrogenation or via the formation of an epoxide
intermediate, followed by subsequent loss of water (21). In this
study, we utilized recombinant CYP3A4, 18O-incorporation
studies, and chemically oxidized raloxifene to elucidate the
mechanisms of CYP3A4-mediated metabolism of raloxifene.
Raloxifene incubated with reconstituted CYP3A4 and termi-
nated with TCA produced two hydroxyraloxifene metabolites and
several GSH adducts (Figure 1). The hydroxyraloxifene metabo-
lites were identified as 7-OHRA and 30-OHRA. One mono-GSH
adduct, GS-RA, was identified by LC/MS/MS analysis. Previous
studiesidentifieduptothreemono-GSHraloxifeneadducts(20,21).
However, those studies used rat and human liver microsomes
which contained a multitude of P450 enzymes, which could
potentially produce additional mono-GSH adducts. It was pos-
sible that multiple mono-GSH adducts coeluted in our analysis,
but careful examination of the products using several highly
varied HPLC conditions did not indicate the presence of addi-
tional adducts (data not shown). Two peaks corresponding to
GS-OHRA metabolites and one peak corresponding to di-GS-
OHRA were produced by CYP3A4 and detected by LC/MS/MS.
Inclusion of the 7-OHRA and 30-OHRA standards in the
CYP3A4 incubation confirmed that the GS-OHRA-1 adduct
was hydroxylated at the 30-position. The site of hydroxylation on
the remaining GS-OHRA and di-GS-OHRA adducts could not
be unequivocally identified.
oxygen or water. Therefore, 7-OHRA was not formed through
CYP3A4-mediated oxygenation or through hydration of the
diquinone methide. GS-RA did not incorporate oxygen from
either molecular oxygen or water, and these results would be
expected if the GS-RA adduct was formed directly by GSH
addition to the diquinone methide intermediate. Alternatively, if
GS-RA was formed via CYP3A4-mediated epoxidation of the
C6-C7 double bond to an arene oxide intermediate, the nucleo-
philic attack at C7 by GSH would result in the formation of a
gem-diol (21) (Scheme 1). Subsequent dehydration of the un-
stable gem-diol could produce the GS-RA metabolite. However,
because there is an equal statistical probability that either of the
gem-diol oxygens would be lost from dehydration, if the arene
oxide was formed at the C6-C7 position, one would expect 50%
of the 6-hydroxyl oxygen to contain 18O incorporated from 18O2.
There was no detectable 18O incorporated into the GS-RA
adduct. Therefore, these results strongly suggest that raloxifene
diquinone methide was directly produced by CYP3A4 and it was
the precursor of GS-RA.
Additional experiments investigated the origins of 7-OHRA. If
7-OHRA production required P450 turnover, but did not obtain
its oxygen atom from either O2 or H2O, what was the source of
the oxygen? Incubations in the presence of catalase or superoxide
dismutase had no effect on 7-OHRA production, and incuba-
tions of raloxifene in the presence of lipid hydroperoxides and
H2O2 failed to produce 7-OHRA (data not shown). Interestingly,
it was noted that when incubations of raloxifene with CYP3A4
were terminated with methanol, all of the raloxifene metabolites
and adducts were observed, with the exception of 7-OHRA. Only
when the incubations were terminated with TCA, or other acids
such as perchloric acid (data not shown), was 7-OHRA formed.
Furthermore, when acid was added to the protein pellet resulting
from methanol quenching and extraction, we could recover
appreciable amounts of 7-OHRA. GSH has previously been
shown to decrease, but not prevent, the irreversible inhibition of
CYP3A4 via raloxifene (21). Our work demonstrated that while
GSH had no effect on 30-OHRA production, the presence of
To establish the mechanism of CYP3A4-mediated oxygena-
tion versus dehydrogenation of raloxifene, 18O-incorporation
studies were utilized to track the source of oxygen in the
raloxifene metabolites. Data indicated that 30-OHRA obtained
greater than 99% of its 30-oxygen from molecular oxygen (i.e.,
through P450-mediated oxygenation). Surprisingly, 7-OHRA
did not incorporate its oxygen atom from either molecular

Article
Biochemistry, Vol. 49, No. 21, 2010 4473
Scheme 2: Proposed Pathway of Raloxifene Oxidation by CYP3A4a
aRCOO- = carboxylic acid residue on CYP3A4 or alternative protein; GSH = glutathione.
and forms an ester with carboxyl groups on surrounding proteins
GSH significantly reduced 7-OHRA production. Together, these
results suggest that the reactive intermediate that inactivates
CYP3A4 is also the reactive intermediate required for 7-OHRA
production. Previous work by Yukinaga et al. (25) showed that
raloxifene formed an adduct with the OH group of tyrosine (Tyr)
75 on the CYP3A4 apoprotein. However, the resulting ether
bond would be extremely difficult to hydrolyze with any aqueous
acidic condition. Therefore, we theorized that the raloxifene
diquinone methide reacted with carboxyl groups on CYP3A4
or other proteins in the reconstituted system (i.e., oxidoreductase
and b5) to produce an ester, which could be hydrolyzed by acid to
release 7-OHRA. A similar unusual mechanism was observed in
the P450-mediated metabolism of benzo[a]pyrene (BaP) to its
diol epoxide, which covalently modified the Asp47 carboxylic
side chain of human hemoglobin to form an ester (34, 35).
Subsequent hydrolysis of the ester formed the alcohol, BaP
7,8,9,10-tetrahydrotetrol.
Due to the reactivity and short half-life of the diquinone
methide (<1 s) (20), it was unlikely that it would travel far
from the CYP3A4 active site, suggesting CYP3A4 was the most
likely site of ester formation. However, the previously reported
raloxifene/CYP3A4 adducts on Cys239, which is adjacent to an
exit channel (36), or Tyr75 on the solvent-exposed surface of
CYP3A4 (observed from the crystal structure) demonstrated that
the diquinone methide was stable enough to travel from the active
site and alkylate these remote nucleophilic residues. Furthermore,
when raloxifene was linked to biotin and then metabolized with
rat liver microsomal incubations, the raloxifene congener still
formed covalent adducts with microsomal proteins that were not
P450s (22). Thus, the diquinone methide must have been stable
enough to exist outside the active site long enough to diffuse and
react with nucleophiles on a protein that was different from the
original bioactivation P450 enzyme. Therefore, we cannot exclude
a mechanism where the diquinone methide leaves the active site
in the reconstituted system (i.e., oxidoreductase and b5).
To test the theory of ester formation, silver oxide was used
to chemically produce raloxifene diquinone methide, which
was trapped with GSH to produce GS-RA, verifying that the
diquinone methide was formed by oxidation of raloxifene. Acetic
acid and propionic acid were employed instead of GSH as
trapping agents to mimic protein carboxylic acid moieties and
used to trap the diquinone methide. LC/MS analysis of these
products detected molecules with [M þ H]þ = m/z 532 and m/z
546, the precise [M þ H]þ ions predicted for raloxifene con-
jugated with acetic acid and propionic acid, respectively. Further-
more, when TCA was added to these products, 7-OHRA was
recovered, strongly suggesting that 7-OHRA was not produced
directly via CYP3A4-mediated oxygenation. Rather, CYP3A4
dehydrogenates raloxifene to a reactive diquinone methide,
which was subsequently trapped by a carboxylic acid functional
side chain on CYP3A4 or a nearby protein, forming an ester.
7-OHRA was released only by the hydrolysis of the ester under
acidic conditions and therefore was not present during the
incubation.
Altogether, the results of this study change our current
understanding of CYP3A4-mediated metabolism of raloxifene
(Scheme 2). This study demonstrated that 30-OHRA appears to
be the only hydroxylated metabolite produced via CYP3A4-
mediated oxygenation. Furthermore, lack of 18O incorporation
from molecular oxygen demonstrates that GS-RA must be
produced from CYP3A4-mediated dehydrogenation of raloxi-
fene to a diquinone methide intermediate, not an arene oxide
intermediate (Scheme 1). Unfortunately, due to the lack of GS-
RA standards, we could not quantify the production of GS-RA.
However, due to the relatively abundant production of 7-OHRA
and GS-RA, dehydrogenation appears to be a major pathway of
raloxifene metabolism.

4474 Biochemistry, Vol. 49, No. 21, 2010
Moore et al.
In contrast, 7-OHRA was not a “normal” metabolite of
CYP3A4. Rather, we propose a highly unusual pathway, in
which raloxifene diquinone methide conjugates with carboxyl
groups of CYP3A4 or nearby proteins to form an ester bond.
Interestingly, this ester bond only forms on the benzothiophene
moiety and not the phenol moiety. Acidic conditions are
required to hydrolyze this ester bond to release 7-OHRA. There-
fore, if 7-OHRA is not produced during the incubation of
raloxifene with CYP3A4, the secondary metabolites (i.e., GS-
OHRA and di-GS-OHRA) are most likely formed by additional
metabolism of 30-OHRA. In support of this rationale, greater
than 99% of the di-GS-OHRA metabolite incorporated oxygen
from molecular oxygen, suggesting it is efficiently formed from
30-OHRA. Unfortunately, due to the lower levels of GS-OHRA
metabolites, we could not determine the source of oxygen in
these metabolites. However, there remains the possibility that
GS-RA is hydroxylated by CYP3A4 to form the GS-OHRA and
di-GS-OHRA products. Additional study is required to deter-
mine the fate of GS-RA and the source of the secondary
metabolites.
In summary, CYP3A4 oxygenates raloxifene to 30-OHRA
and dehydrogenates raloxifene to a reactive diquinone methide.
The reactive species can be trapped by GSH in the form of
thioether conjugates, form adducts with Cys239 and Tyr75 of
CYP3A4, or, in a novel mechanism, conjugate with carboxyl
groups, forming ester conjugates with CYP3A4 or nearby
proteins. Furthermore, these results suggest that the benzothio-
phene moiety, and not the phenol moiety, of raloxifene is the
structural feature responsible for formation of the reactive
species. These findings support the rational design of new
SERMs without the benzothiophene structure to reduce bioac-
tivation liabilities. Other putative heterocyclic functional groups
that could be “structural alerts” include the benzofuran, indole,
and benzimidazole moieties.
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ACKNOWLEDGMENT
21. Chen, Q., Ngui, J. S., Doss, G. A., Wang, R. W., Cai, X., DiNinno,
F. P., Blizzard, T. A., Hammond, M. L., Stearns, R. A., Evans, D. C.,
Baillie, T. A., and Tang, W. (2002) Cytochrome P450 3A4-mediated
bioactivation of raloxifene: Irreversible enzyme inhibition and thiol
adduct formation. Chem. Res. Toxicol. 15, 907–914.
The authors thank Dr. Judy Bolton, University of Illinois,
Chicago, for providing 30-hydroxyraloxifene and 7-hydroxyral-
oxifene for these studies.
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