4-Hydroxylated metabolites of the antiestrogens tamoxifen and toremifene are metabolized to unusually stable quinone methides

Article abstract of DOI:10.1021/tx990144v

Tamoxifen is widely prescribed for the treatment of hormone-dependent breast cancer, and it has recently been approved by the Food and Drug Administration for the chemoprevention of this disease. However, long-term usage of tamoxifen has been linked to increased risk of developing endometrial cancer in women. One of the suggested pathways leading to the potential toxicity of tamoxifen involves its oxidative metabolism to 4- hydroxytamoxifen, which may be further oxidized to an electrophilic quinone methide. The resulting quinone methide has the potential to alkylate DNA and may initiate the carcinogenic process. To further probe the chemical reactivity and toxicity of such an electrophilic species, we have prepared the 4-hydroxytamoxifen quinone methide chemically and enzymatically, examined its reactivity under physiological conditions, and quantified its reactivity with GSH. Interestingly, this quinone methide is unusually stable; its half- life under physiological conditions is approximately 3 h, and its half-life in the presence of GSH is approximately 4 min. The reaction between 4- hydroxytamoxifen quinone methide and GSH appears to be a reversible process because the quinone methide GSH conjugates slowly decompose over time, regenerating the quinone methide as indicated by LC/MS/MS data. The tamoxifen GSH conjugates were detected in microsomal incubations with 4- hydroxytamoxifen; however, none were observed in breast cancer cell lines (MCF-7) perhaps because very little quinone methides is formed. Toremifene, which is a chlorinated analogue of tamoxifen, undergoes similar oxidative metabolism to give 4-hydroxytoremifene, which is further oxidized to the corresponding quinone methide. The toremifene quinone methide has a half-life of approximately 1 h under physiological conditions, and its rate of reaction in the presence of excess GSH is approximately 6 min. More detailed analyses have indicated that the 4-hydroxytoremifene quinone methide reacts with two molecules of GSH and loses chlorine to give the corresponding di-GSH conjugates. The reaction mechanism likely involves an episulfonium ion intermediate which may contribute to the potential cytotoxic effects of toremifene. Similar to what was observed with 4-hydroxytamoxifen, 4- hydroxytoremifene was metabolized to di-GSH conjugates in microsomal incubations at about 3 times the rate of 4-hydroxytamoxifen, although no conjugates were detected with MCF-7 cells. Finally, these data suggest that quinone methide formation may not make a significant contribution to the cytotoxic and genotoxic effects of tamoxifen and toremifene.

Full text of DOI:10.1021/tx990144v

Chem. Res. Toxicol. 2000, 13, 45-52
45
4-Hyd r oxyla ted Meta bolites of th e An tiestr ogen s
Ta m oxifen a n d Tor em ifen e Ar e Meta bolized to
Un u su a lly Sta ble Qu in on e Meth id es
Peter W. Fan, Fagen Zhang, and J udy L. Bolton*
Department of Medicinal Chemistry and Pharmacognosy (M/ C 781), College of Pharmacy,
University of Illinois at Chicago, 833 South Wood Street, Chicago, Illinois 60612-7231
Received August 3, 1999
Tamoxifen is widely prescribed for the treatment of hormone-dependent breast cancer, and
it has recently been approved by the Food and Drug Administration for the chemoprevention
of this disease. However, long-term usage of tamoxifen has been linked to increased risk of
developing endometrial cancer in women. One of the suggested pathways leading to the
potential toxicity of tamoxifen involves its oxidative metabolism to 4-hydroxytamoxifen, which
may be further oxidized to an electrophilic quinone methide. The resulting quinone methide
has the potential to alkylate DNA and may initiate the carcinogenic process. To further probe
the chemical reactivity and toxicity of such an electrophilic species, we have prepared the
4-hydroxytamoxifen quinone methide chemically and enzymatically, examined its reactivity
under physiological conditions, and quantified its reactivity with GSH. Interestingly, this
quinone methide is unusually stable; its half-life under physiological conditions is approximately
3 h, and its half-life in the presence of GSH is approximately 4 min. The reaction between
4-hydroxytamoxifen quinone methide and GSH appears to be a reversible process because the
quinone methide GSH conjugates slowly decompose over time, regenerating the quinone
methide as indicated by LC/MS/MS data. The tamoxifen GSH conjugates were detected in
microsomal incubations with 4-hydroxytamoxifen; however, none were observed in breast cancer
cell lines (MCF-7) perhaps because very little quinone methides is formed. Toremifene, which
is a chlorinated analogue of tamoxifen, undergoes similar oxidative metabolism to give
4-hydroxytoremifene, which is further oxidized to the corresponding quinone methide. The
toremifene quinone methide has a half-life of approximately 1 h under physiological conditions,
and its rate of reaction in the presence of excess GSH is approximately 6 min. More detailed
analyses have indicated that the 4-hydroxytoremifene quinone methide reacts with two
molecules of GSH and loses chlorine to give the corresponding di-GSH conjugates. The reaction
mechanism likely involves an episulfonium ion intermediate which may contribute to the
potential cytotoxic effects of toremifene. Similar to what was observed with 4-hydroxytamoxifen,
4-hydroxytoremifene was metabolized to di-GSH conjugates in microsomal incubations at about
3 times the rate of 4-hydroxytamoxifen, although no conjugates were detected with MCF-7
cells. Finally, these data suggest that quinone methide formation may not make a significant
contribution to the cytotoxic and genotoxic effects of tamoxifen and toremifene.
(15-17), o-quinone (18, 19), and quinone methide (20-
22). The mechanism of formation of the quinone methide
likely involves P450-catalyzed aromatic hydroxylation of
tamoxifen generating 4-hydroxytamoxifen, which under-
goes two-electron oxidation of the π system resulting in
the quinone methide (Scheme 1) (23-26).
Recently, it has been shown that the 4-hydroxytamox-
ifen quinone methide reacts with DNA to form covalent
adducts in vitro (22). However, very little is known about
the reactivity, rate of formation, and other potential
biological targets of this electrophile. To further probe
the chemical reactivity and toxicity of the tamoxifen
quinone methide, we have synthesized it chemically and
enzymatically, measured its half-life in solution, quanti-
fied its reactivity with GSH,1 and characterized the GSH
conjugates. Interestingly, we have found that very little
quinone methide was formed in microsomal incubations
with 4-hydroxytamoxifen. In addition, the reaction be-
tween this quinone methide and GSH may be a reversible
In tr od u ction
With recent approval of tamoxifen (Nolvadex) for the
prevention of breast cancer by the Food and Drug
Administration, a deeper understanding of the risk
factors involved with long-term usage of this drug is
crucial. Despite the beneficial effects of tamoxifen for
women who are at risk of developing breast cancer,
reports have shown that tamoxifen may increase the
incidence of endometrial cancer in women (1-7), may
induce the formation of DNA adducts (8-10), and causes
hepatocarcinoma in animal models (11-14). Several
bioactivation pathways leading to the toxicity of tamox-
ifen have been proposed. They include the generation of
reactive intermediates such as the tamoxifen carbocation
* To whom correspondence should be addressed: Department of
Medicinal Chemistry and Pharmacognosy (M/C 781), College of
Pharmacy, University of Illinois at Chicago, 833 S. Wood St., Chicago,
IL 60612-7231. Telephone: (312) 996-5280. Fax: (312) 996-7107.
E-mail: judy.bolton@uic.edu.
10.1021/tx990144v CCC: $19.00 © 2000 American Chemical Society
Published on Web 12/18/1999

46 Chem. Res. Toxicol., Vol. 13, No. 1, 2000
Fan et al.
Sch em e 1. Biotr a n sfor m a tion of Ta m oxifen a n d Tor em ifen e to th e Cor r esp on d in g Qu in on e Meth id es
of the HPLC effluent, and a nitrogen bath gas at 300 °C and a
flow rate of 10 L/min were used for evaporation of the solvent
from the electrospray. A mass range from 300 to 1050 mass
units was scanned every 1.5 s. Tandem MS (MS/MS) spectra
were obtained using a Micromass (Manchester, U.K.) Quattro
II triple-quadrupole mass spectrometer equipped with an elec-
trospray ionization source and a Hewlett-Packard 1050 HPLC
system. Collision-induced dissociation (CID) was carried out
using a range of collision energy from 25 to 70 eV and argon
collision gas pressure of 2.7 µbar.
HP LC Met h od ology. Two general methods were used to
analyze and separate the quinone methides and the GSH
conjugates. All retention times reported in the text were
obtained using method A.
Meth od A. Analytical HPLC analysis was performed using
a 4.6 mm × 150 mm Ultrasphere C-18 column (Beckman) on
the above-mentioned Shimadzu HPLC system. The mobile phase
consisted of 20% methanol in 0.25% perchloric acid/0.25% acetic
acid (pH 3.5) with a flow rate of 1.0 mL/min for 5 min, which
was increased to 35% CH3OH over the course of 1 min, then to
60% over the course of the next 39 min, and finally to 95% CH3-
OH over the course of the last 5 min.
Meth od B. LC/MS analysis was performed using a 4.6 mm
× 150 mm Ultrasphere C-18 column (Beckman) on the above-
mentioned Hewlett-Packard LC/MS system, and tandem mass
spectra were obtained using the Micromass system interfaced
with the Hewlett-Packard HPLC system. The mobile phase
consisted of 20% methanol in 0.5% ammonium acetate (pH 3.5)
with a flow rate of 1.0 mL/min for 5 min, which was increased
to 35% CH3OH over the course of 1 min, then to 60% over the
course of the next 39 min, and finally to 95% CH3OH over the
course of the last 5 min.
P r ep a r a tion of th e Ta m oxifen a n d Tor em ifen e Qu in on e
Meth id es. Typically, (Z)-4-hydroxytamoxifen or (Z)-4-hydroxy-
toremifene (2 mg) was dissolved in acetone (1 mL) in the dark
and cooled to 0-5 °C. Upon careful addition of freshly prepared
manganese dioxide [20 mg (22)], the suspension was stirred for
1 h and centrifuged. The yellow supernatant containing 4-hy-
droxytamoxifen quinone methide or 4-hydroxytoremifene quino-
ne methide was removed and concentrated to a final volume of
500 µL under N2. An aliquot (25 µL) of the supernatant was
immediately analyzed by LC/MS (method B). 4-Hyd r oxyta -
m oxifen qu in on e m eth id e: UV (CH3CN) 200, 230, 280, 400
nm; positive ion electrospray MS m/z (rel intensity) 386 (100%)
[M + H]+; MS/MS of m/z 386 (rel intensity) 341 (20%), 314 (10%),
221 (13%), 128 (5%), and 72 (100%); retention time of 52 min.
4-Hydroxytoremifene quinone methide was prepared similarly
from (Z)-4-hydroxytoremifene and analyzed in a similar manner.
4-Hyd r oxytor em ifen e qu in on e m eth id e: UV (CH3CN) 208,
238, 302, 382 nm; positive ion electrospray MS m/z (rel intensity)
420 (100%) [M + H]+.
process because its GSH conjugates slowly decompose
over time, regenerating the quinone methide. As a result,
quinone methide formation from tamoxifen may not play
a major role in the carcinogenic effects attributed to the
parent compound.
Toremifene is a chlorinated structural analogue of
tamoxifen. It was introduced in the late 1980s for the
treatment of advanced stages of breast cancer (27).
4-Hydroxytoremifene, which may play a role similar to
that of 4-hydroxytamoxifen (28), is one of the metabolites
of toremifene (29) which could be further oxidized to an
electrophilic quinone methide. Therefore, we have inves-
tigated the rate of formation of the 4-hydroxytoremifene
quinone methide in microsomal incubations, measured
its half-life under physiological conditions, and examined
the conjugates formed with GSH. Unlike the tamoxifen
quinone methide, the toremifene quinone methide forms
di-GSH conjugates through a mechanism which likely
involves an additional electrophilic intermediate, the
episulfonium ion. It is possible that episulfonium ion
formation contributes to the biological effects of toremifene.
Ma ter ia ls a n d Meth od s
Ca u tion : The quinone methides were handled in accordance
with NIH guidelines for the Laboratory Use of Chemical
Carcinogens (30). All chemicals were purchased from Aldrich
(Milwaukee, WI), Fisher Scientific (Itasca, IL), or Sigma (St.
Louis, MO) unless stated otherwise. [3H]GSH (glycine-2-3H) was
obtained from Dupont (Boston, MA) and diluted to a specific
activity of 40 nCi/nmol. The biologically active isomers (Z)-4-
hydroxytamoxifen and (Z)-4-hydroxytoremifene were stereose-
lectively synthesized by the McMurry reaction as described
previously (31).
In str u m en ta tion . HPLC experiments were performed on a
Shimadzu LC-10A gradient HPLC system equipped with an
SIL-10A autoinjector, an SPD-M10AV UV/vis photodiode array
detector, and an SPD-10AV detector. Peaks were integrated
with Shimadzu EZ-Chrom software and a 486 personal com-
puter. UV spectra were measured on a Hewlett-Packard 8452A
photodiode array UV/vis spectrophotometer. 1H NMR spectra
were obtained with a Bruker Avance DPX300 spectrometer at
300 MHz. Positive ion electrospray mass spectra were obtained
using a Hewlett-Packard 5989B engine quadrupole mass spec-
trometer equipped with a ChemStation data system and high-
flow pneumatic nebulizer-assisted electrospray LC/MS interface.
The mass spectrometer was interfaced with a Hewlett-Packard
(Palo Alto, CA) 1050L gradient HPLC system equipped with a
photodiode array UV/vis absorbance detector set at 230-450
nm. The temperature of the quadrupole analyzer was main-
tained at 120 °C, and unit resolution was used for all measure-
ments. Nitrogen at a pressure of 80 psi was used for nebulization
Kin etic Exp er im en ts. The disappearance of either 4-hy-
droxytamoxifen quinone methide or 4-hydroxytoremifene quino-
ne methide (0.16 mM) in 50 mM K2HPO4 buffer (10 mL, pH
7.4, 37 °C) was followed by monitoring the decrease in UV
absorbance at 400 nm (5 min/scan) using a Hewlett-Packard
8452A diode array spectrophotometer. Initially, an aliquot (1
mL) of the reaction solution was removed, and its absorbance
was scanned. Aliquots (1 mL) were then removed every 4 min
and centrifuged at 13 000 rpm for 30 s to remove any precipitate
1
Abbreviations: 4-OHTAMQM, 4-hydroxytamoxifen quinone me-
thide; 4-OHTORQM, 4-hydroxytoremifene quinone methide; GSH,
glutathione; 4-OHTAM-SG, glutathione conjugates of 4-hydroxyta-
moxifen; 3,4-di-OHTAM-SG, glutathione conjugates of 3,4-dihydrox-
ytamoxifen; 4-OHTOR-diSG, disubstituted glutathione conjugates of
4-hydroxytoremifene.

Quinone Methides of Tamoxifen and Toremifene
Chem. Res. Toxicol., Vol. 13, No. 1, 2000 47
Ta ble 1. Rea ctivity of th e Ta m oxifen a n d Tor em ifen e
Qu in on e Meth id esa
activity of 40 nCi/nmol) was added in phosphate buffer, to
achieve final concentrations of 0.5 and 2.5 mM, respectively.
An NADPH-generating system consisting of 1 mM NADP+, 5
mM isocitric acid, and 0.2 unit/mL isocitric acid dehydrogenase
was used together with 5.0 mM MgCl2. For control incubations,
NADP+ was omitted. The reactions were initiated by the
addition of NADP+ and terminated by chilling in an ice bath
followed by the addition of perchloric acid (25 µL).
Ad d u ct Qu a n tifica tion . The incubates were centrifuged at
13 000 rpm for 5 min to precipitate microsomal protein. Aliquots
of the supernatant (100 µL) were analyzed directly by HPLC
(method A). For quantification of GSH conjugates, aliquots (300
µL) of the column effluent were collected during each run, and
radioactivity was measured with a Beckman model LS 5801
liquid scintillation counter. Concentrations of the GSH conju-
gates were calculated by adding the radioactivities associated
with each peak and converting the data to nanomolar amounts
using the specific activity of [3H]GSH (Table 2).
Dir ect Detection of th e Ta m oxifen Qu in on e Meth id e in
Micr osom a l In cu ba tion s. In another set of experiments,
4-hydroxytamoxifen was incubated with rat liver microsomes
using the same incubation conditions described above except
GSH was omitted and the total incubation volume was 10 mL.
A different workup procedure was adopted to preserve 4-hy-
droxytamoxifen quinone methide generated from microsomal
incubation. The incubates were saturated with NaCl and
extracted with ethyl acetate (2 × 10 mL). The combined ethyl
acetate layers were concentrated to 500 µL under nitrogen and
diluted with 1 mL of acetonitrile. An aliquot (25 µL) was then
analyzed using LC/MS (method B).
In cu ba tion of 4-Hyd r oxyta m oxifen a n d 4-Hyd r oxy-
tor em ifen e in MCF -7 Cells. Briefly, MCF-7 cells were main-
tained in MEME supplemented with 1% penicillin, 10 mg/L
streptomycin, 10 mg/L fungizome, 1% nonessential amino acids,
and 10% bovine serum. The cells were harvested by trypsiniza-
tion, counted, and diluted to a density of ∼106 cells/mL of
medium. Aliquots (10 mL) of the diluted cells were transferred
to three pasteurized incubation tubes. 4-Hydroxytamoxifen (or
4-hydroxytoremifene) was added to yield final concentrations
of 0, 20, and 50 µM. DMSO was used as the control. The cells
were then incubated at 37 °C for 30 min. Incubations were
terminated by chilling the tubes containing the cells in an ice
bath followed by addition of perchloric acid (50 µL/mL) to each
tube. The incubates were centrifuged at 13 000 rpm for 6 min
to precipitate cellular debris. After centrifugation, the super-
natants were extracted using C18 solid-phase extraction car-
tridges (Oasis; HLB Cartridge, Waters Corp., Milford, MA) and
eluted with methanol. The methanol was concentrated to a final
volume of 300 µL, and aliquots (150 µL) were analyzed directly
by HPLC using method A.
phosphate buffer
half-life (min)
50 mM
GSH half-life (min)
substrate
4-OHTAMQM
4-OHTORQM
174 ( 41
57 ( 8
3.9 ( 0.1
5.7 ( 0.3
a
The rates of disappearance of quinone methides at 410 nm as
described in Materials and Methods (pH 7.4, 37 °C). Results are
the means ( SD of three determinations.
before recording the absorbance. Finally, an aliquot of the
quinone methide solution in buffer was left in the quartz cuvette,
and its rate of decomposition at 410 nm was continuously
monitored at the same scan rate. Rate constants were deter-
mined in triplicate for at least six half-lives (Table 1). The
disappearance of either 4-hydroxytamoxifen quinone methide
(0.16 mM) or 4-hydroxytoremifene quinone methide (0.14 mM)
in the presence of GSH (50 mM) in phosphate buffer (50 mM,
pH 7.4) was followed by monitoring the decrease in absorbance
at 400 nm (10 s/scan) at 37 °C. Rate constants were determined
as described above (Table 1).
GSH Con ju ga tes of th e Ta m oxifen a n d Tor em ifen e
Qu in on e Met h id es. GSH conjugates of 4-hydroxytamoxifen
quinone methide were prepared by incubating the quinone
methide (1.6 mM) with 50 mM GSH in 50 mL of K2HPO4 buffer
(50 mM, pH 7.4) at 37 °C for 30 min. The reaction was
terminated by extracting the aqueous solution with 50 mL of
ether. The GSH conjugates were isolated from the aqueous
phase on PrepSep C18 extraction cartridges (Fisher Scientific,
Fair Lawn, NJ ) and eluted with methanol. The methanol was
concentrated to a final volume of 200 µL, and an aliquot (25
µL) was analyzed using LC/MS (method B). 4-OHTAM-SG 1:
UV (CH3OH) 245, 275 nm; positive ion electrospray MS m/z (rel
intensity) 693 (100%) [M + H]+; retention time of 25 min.
4-OHTAM-SG 2 a n d 3 (u n r esolved ): UV (CH3OH) 246, 275
nm; positive ion electrospray MS m/z (rel intensity) 693 (100%)
[M + H]+; retention time of 28 min. 4-OHTAM-SG 4: UV (CH3-
1
OH) 246, 275 nm; H NMR (CD3OD) δ 1.18 (t, 3H, CH3), 1.88
(m, 2H, Gluâ), 2.45 (m, 2H, Gluγ), 2.88 (m, 1H, Cysâ), 2.94 [s,
6H, N(CH3)2], 3.29 (m, 1H, Cysâ), 3.59 (t, 2H, CH2N), 3.59 (m,
1H, GluR), 3.76 (d, 2H, GlyR), 4.33 (m, 1H, CysR), 4.60 (m, 1H,
CH3H) 4.68 (t, 2H, OCH2), 6.65 (d, 2H, J ) 5.2 Hz, OPhH), 6.99
(d, 2H, J ) 5.2 Hz, OPhH), 7.07 (d, 2H, J ) 5.2 Hz, OPhH),
7.23-7.66 (7H, m, ArH); positive ion electrospray MS m/z (rel
intensity) 693 (100%) [M + H]+; MS/MS of m/z 693 (rel intensity)
m/z 564 (30%) [MH - Glu]+, 386 (100%) [MH - GSH]+, 341
(10%) [MH - GSH - HN(CH3)2]+; retention time of 30 min. A
similar procedure was used to generate the 4-hydroxytoremifene
quinone methide GSH conjugates. 4-OHTOR -d iSG 1 a n d 2
(p a r tia lly r esolved ): UV (CH3OH) 250, 280 nm; positive ion
electrospray MS m/z (rel intensity) 998 (15%) [M + H]+, 500
(100%) [M + 2H]2+; MS/MS of m/z 998 (major ions) (rel intensity)
m/z 869 (10%) [MH - Glu]+, 691 (95%) [MH - GSH]+, 673 (30%)
[MH - GSH - H2O]+, 562 (60%) [MH - GSH - Glu]+; retention
time of 13 min. 4-OHTOR-d iSG 3 a n d 4 (p a r tia lly r esolved ):
UV (CH3OH) 245, 280 nm; positive ion electrospray MS m/z (rel
intensity) 998 (15%) [M + H]+, 500 (100%) [M + 2H]2+; MS/MS
of m/z 998 (rel intensity) m/z 869 (10%) [MH - Glu]+, 850 (12%)
[MH - Glu - H2O]+, 691 (30%) [MH - GSH]+, 673 (20%) [MH
- GSH - H2O]+, 562 (50%) [MH - GSH - Glu]+; retention time
of 16 min.
In cu ba tion s. Female Sprague-Dawley rats (180-200 g) were
obtained from Sasco Inc. (Omaha, NE). The rats were pretreated
with dexamethasone to induce P450 3A isozymes. They were
given dexamethasone in corn oil (100 mg/kg) daily via ip
injection for 3 days and sacrificed on day 4. Protein and P450
concentrations of the liver microsomes were determined as
described by Thompson et al. (32). Incubations containing
microsomal protein (1 nmol of P450/mL) were conducted for 15
min at 37 °C in 50 mM phosphate buffer (pH 7.4, 0.5 mL total
volume). Substrates (4-hydroxytamoxifen or 4-hydroxytoremifene)
were added as solutions in DMSO, and [3H]GSH (specific
Resu lts a n d Discu ssion
Ch em ica l Gen er a t ion of t h e Ta m oxifen a n d
Tor em ifen e Qu in on e Meth id es. Mild oxidants such as
silver(I) oxide (22, 33), lead(IV) oxide (34, 35), and
manganese(IV) oxide (36) have been reported for the
efficient synthesis of quinones and quinone methides
from various phenolic compounds. In this study, we found
that activated γ-manganese dioxide served as the most
effective oxidant for 4-hydroxytamoxifen (22). The UV
spectra exhibited a strong absorbance around 400 nm,
which was absent in the starting material and was
characteristic of most quinones and quinone methides
(34, 35). LC/MS analysis revealed the appearance of a
new peak with a retention time of 52 min and a
protonated molecular ion at m/z 386. MS/MS analysis of
m/z 386 with CID gave the fragment ion spectra shown
in Figure 1, which strongly suggested that the new peak
was 4-hydroxytamoxifen quinone methide. Similar oxida-
tion experiments with 4-hydroxytoremifene gave the

48 Chem. Res. Toxicol., Vol. 13, No. 1, 2000
Fan et al.
Ta ble 2. Con ver sion of 4-Hyd r oxyta m oxifen a n d 4-Hyd r oxytor em ifen e to th e GSH Con ju ga tes by Ra t Liver Micr osom esa
substrate
conjugate
retention time (min)
rate of formation [nmol (nmol of P450)-1 (10 min)-1
]
4-hydroxytamoxifen
quinone methide
4-OHTAM-SG 1
25
28
30
0.0064 ( 0.0008
0.011 ( 0.003
0.0058 ( 0.0014
total: 0.023 ( 0.0052
0.0069 ( 0.002
0.0047 ( 0.0002
0.0037 ( 0.0002
total: 0.015 ( 0.0024
4-OHTAM-SG 2 and 3
4-OHTAM-SG 4
o-quinone
3,4-di-OHTAM-SG 1
3,4-di-OHTAM-SG 2
3,4-di-OHTAM-SG 3
34
37
41
4-hydroxytoremifene
quinone methide
4-OHTOR-diSG 1 and 2
4-OHTOR-diSG 3 and 4
13
16
0.06 ( 0.018
0.031 ( 0.006
total: 0.091 ( 0.024
a
Incubations were conducted for 15 min with 0.5 mM substrate, 2.5 mM [3H]GSH (specific activity of 40 nCi/nmol), liver microsomes
from dexamethasone-induced female rats (1 nmol of P450/mL), and an NADPH-generating system. Results are the means ( SD of three
determinations.
F igu r e 2. Kinetic studies on the reaction of 4-hydroxytamox-
ifen quinone methide with GSH. Incubations contained 4-hy-
droxytamoxifen (0.16 mM) and GSH (50 mM) at pH 7.4 and 37
°C. UV scans were over the 200-600 nm wavelength range, at
a rate of 10 s/scan.
F igu r e 1. MS/MS fragmentation pattern of 4-hydroxytamox-
ifen quinone methide.
Sch em e 2. Rea ction of 4-Hyd r oxytor em ifen e
Qu in on e Meth id e w ith GSH w ith a n In itia l
1,8-Mich a el Ad d ition of On e Molecu le of GSH to
expected protonated molecule at m/z 420 in the positive
ion electrospray mass spectrum. Furthermore, the solu-
tion containing 4-hydroxytoremifene quinone methide
exhibited a strong UV absorbance around 400 nm as
expected for quinone methide generation.
th e Qu in on e Meth id e F ollow ed by th e F or m a tion
of a n Ep isu lfon iu m Ion Wh ich Rea cts w ith a
Secon d GSH Molecu le To Give a Disu bstitu ted
GSH Con ju ga te
Rea ctivity of th e Ta m oxifen a n d Tor em ifen e
Qu in on e Meth id es. There have been some reports in
the literature about the chemistry and reactivity of
p-quinone methides (37, 38). They are known for their
electrophilicity and low capacity for redox chemistry (39).
To explore the reactivity of tamoxifen or toremifene
quinone methides, we measured their half-lives under
physiological conditions and in the presence of excess
GSH. Interestingly, the quinone methide derived from
4-hydroxytamoxifen is unusually stable; its half-life
under physiological conditions was approximately 3 h,
and its half-life in the presence of excess GSH was
approximately 4 min (Figure 2 and Table 1). The unusual
stability is readily explained by the contribution of an
extensive resonance stabilization (conjugated π system)
to the overall resonance hybrid of the quinone methide.
The 4-hydroxytoremifene quinone methide had a half-
life of approximately 1 h under physiological conditions,
and its rate of reaction in the presence of excess GSH
was approximately 6 min (Table 1). More detailed
analysis indicated that the 4-hydroxytoremifene quinone
methide reacts with two molecules of GSH and loses
chlorine to give the corresponding di-GSH conjugates as
described below. This substitution reaction may be
facilitated by a three-membered ring sulfonium ion
intermediate (episulfonium ion, Scheme 2). A GSH
molecule first reacts with 4-hydroxytoremifene quinone
methide via 1,8-Michael addition to generate a half-
mustard intermediate (40). The thiol group then displaces
the chlorine and forms an episulfonium ion. Finally, a

Quinone Methides of Tamoxifen and Toremifene
Chem. Res. Toxicol., Vol. 13, No. 1, 2000 49
Sch em e 3. Rea ction of 4-Hyd r oxyta m oxifen
Qu in on e Meth id e w ith GSHa
B
F igu r e 3. HPLC chromatogram of 4-hydroxytamoxifen-GSH
conjugates. 4-OHTAM-SG 2 and 3 were resolved using a
different HPLC method (unpublished results).
second GSH molecule reacts with the episulfonium ion
to give the disubstituted GSH conjugates (Scheme 2).
There have been several reports about the possible
involvement of an episulfonium ion as a reaction inter-
mediate in the reactions of GSH with various known
chemical carcinogens (40-46). As a result, it is possible
that the episulfonium ion contributes to the potential
cytotoxic effects of toremifene.
a
(A) 1,8-Michael addition of GSH to the quinone methide and
(B) 1,6-Michael addition of GSH to the quinone methide.
GSH Con ju ga tes of 4-Hyd r oxyta m oxifen Qu in on e
Meth id e. Glutathione has been shown to be an effective
agent for trapping quinone methides due to the nucleo-
philicity of the cysteine sulfhydryl group and the rela-
tively high concentration of GSH in vivo. We have found
that incubation of the 4-hydroxytamoxifen quinone me-
thide with GSH resulted in the disappearance of the
distinct yellow color, indicating that a reaction had taken
place. An HPLC method for separating and character-
izing the GSH conjugates by UV and electrospray MS
was developed. Four GSH-dependent peaks (4-OHTAM-
SG 1-4, Figure 3) were detected in the HPLC chromato-
gram, two of which were unresolved (peaks 2 and 3). On
the basis of the qualitative comparison between the total
peak area of GSH conjugates formed from the quinone
methide and the total peak area of unreacted 4-hydroxy-
tamoxifen, the chemical oxidation process was only 17%
efficient. Four GSH-dependent peaks were expected since
GSH could react with 4-hydroxytamoxifen quinone me-
thide either through 1,8-Michael addition to give two
isomers of 4-OHTAM-SG adducts (E and Z isomers) or
through 1,6-Michael addition to give two additional
isomers (Scheme 3; 35). We have found that these GSH
adducts are unstable and decompose slowly over time (7
days). As a result, we were only able to partially purify
one of the GSH conjugates (4-OHTAM-SG 4). The 1H
NMR chemical shifts obtained for this conjugate are
given in Materials and Methods. The assignments were
based on a comparison of 1H NMR data for 4-hydroxyta-
moxifen and other tamoxifen-GSH adducts published in
ref 54. However, it was difficult to make specific assign-
ments for all the peaks because of solvent peak interfer-
ence. Nevertheless, the position of the GSH molecule is
likely to be at the R position. The product ion spectra of
the protonated molecule of 4-OHTAM-SG 4 (m/z 693)
gave abundant fragment ions at m/z 564 (30% rel
intensity) [MH - Glu]+, 386 (100% rel intensity) [MH -
GSH]+, and 341 (10% rel intensity) [MH - GSH - HN-
(CH3)2]+. Loss of 129 mass units (pyroglutamic acid) from
the protonated molecule generated the fragment ion at
m/z 564, which is indicative of a typical GSH conjugate
F igu r e 4. MS/MS fragmentation pattern of the 4-hydroxyta-
moxifen-GSH conjugate.
fragmentation pattern in MS/MS (Figure 4) (47-49). The
fragment ion at m/z 341 is generated from the fragment
ion at m/z 386 after losing the dimethyl amino group.
1
After H NMR data had been obtained, LC/MS analysis
of this conjugate showed the appearance of a new peak
(retention time of 52 min) at m/z 386, suggesting that
the quinone methide had been regenerated. Tandem
mass spectrometry of the m/z 386 ion gave the same
fragment ion spectra as the standard 4-hydroxytamoxifen
quinone methide (Figure 1), strongly suggesting that the
new peak is the quinone methide. This also suggests that
the reaction between 4-hydroxytamoxifen and GSH is a
reversible process.
GSH Con ju ga tes of 4-Hyd r oxytor em ifen e Qu in o-
n e Meth id e. In contrast with the tamoxifen quinone
methide, the reaction between 4-hydroxytoremifene quino-
ne methide and GSH resulted in the formation of di-GSH
conjugates. Four GSH-dependent peaks were detected in
the HPLC chromatogram (4-OHTOR-diSG 1-4). They all
gave identical protonated molecules at m/z 998 (15% rel
intensity) [M + H]+. A doubly charged ion at m/z 500 [M
+ 2H]2+ was also observed in higher abundance (100%)
that had the same retention time as the ion at m/z 998.
On the basis of the qualitative comparison between the
total peak area of GSH conjugates formed from the

50 Chem. Res. Toxicol., Vol. 13, No. 1, 2000
Fan et al.
previous studies have indirectly shown that 4-hydroxy-
tamoxifen can be oxidized to the catechol, 3,4-dihydroxy-
tamoxifen, by microsomes from phenobarbital-induced
rats (18, 19). In these studies, 3,4-dihydroxytamoxifen
was not isolated directly but rather the formation of a
catechol metabolite was assumed from analysis of a
monomethylated metabolite using [3H]-S-adenosyl-L-me-
thionine and endogenous catechol-O-methyltransferase
(18). As catechols are highly redox active compounds
which can be converted by virtually any oxidative en-
zyme, metal ion, or, in some cases, molecular oxygen to
o-quinones which react with GSH, it is not surprising
that the GSH conjugates of 3,4-dihydroxytamoxifen-o-
quinone were observed in our experiments. The amount
of o-quinone-derived GSH conjugates that was produced
was considerably lower than that reported using the
methylation to trap the catechol (18), perhaps due to
differences in the inducing agent as well as other varia-
tions in experimental protocols. No additional GSH-
dependent peaks were observed in corresponding incu-
bations with 4-hydroxytoremifene which suggests that
two-electron oxidation to the quinone methide instead of
aromatic hydroxylation and oxidation to an o-quinone is
the preferred bioactivation pathway. However, synthesis
of 3,4-dihydroxytoremifene, the corresponding o-quinone,
and GSH conjugates will be necessary before the o-
quinone pathway can be discounted for 4-hydroxy-
toremifene.
F igu r e 5. MS/MS fragmentation pattern of the 4-hydroxy-
toremifene-di-GSH conjugate.
quinone methide and the total peak area of unreacted
4-hydroxytoremifene, the chemical oxidation was 30%
efficient. Tandem MS showed loss of 129 mass units
(pyroglutamic acid) from the protonated molecule at m/z
998 (Figure 5). In addition, the product ion spectra of the
ion at m/z 998 also showed the loss of one GSH molecule
(m/z 691) and another loss of a pyroglutamic acid moiety
from the second GSH molecule (m/z 562).
In cu b a t ion of 4-Hyd r oxyt a m oxifen a n d 4-Hy-
d r oxytor em ifen e in MCF -7 Cells. Treating a human
breast cancer cell line (MCF-7) containing the estrogen
receptor with antiestrogens provides a better and more
accurate in vitro model system compared to microsomal
incubations. Hence, it was selected as our model system
for investigating whether quinone methide GSH conju-
gates are formed. However, several attempts to detect
by UV the presence of GSH conjugates in MCF-7 cells
treated with either 4-hydroxytamoxifen or 4-hydroxy-
toremifene failed. We have validated our method by
treating the cells with 3,4-dihydroxytamoxifen, another
metabolite of tamoxifen, and its GSH conjugates were
detected (54). This suggests that very little quinone
methide is formed in MCF-7 cells treated with either
4-hydroxytamoxifen or 4-hydroxytoremifene and that the
UV detection method was not as sensitive as the radio-
labeled GSH used in microsomal incubations.
In conclusion, we have shown that the 4-hydroxylated
metabolites of tamoxifen and toremifene are both oxi-
dized to unusually stable quinone methides. These elec-
trophiles do react with GSH to form conjugates; however,
the rate of reaction is relatively slow compared to those
of most p-quinone methides (25), and the conjugates that
are formed are not stable. In fact, the addition of GSH
to the tamoxifen quinone methide appears to be a
reversible reaction which suggests that little GSH deple-
tion resulting in toxicity would occur in vivo. The
toremifene quinone methide forms di-GSH conjugates
likely through a mechanism involving formation of an
episulfonium ion intermediate, and it is possible that this
additional electrophile contributes to the potential toxic-
ity of toremifene in vivo. Finally, we have shown that
very little quinone methide GSH conjugate was detected
from either phenol in microsomal incubations, and none
were observed in MCF-7 cells. These data suggest that
the slow rate of formation of the quinone methides from
tamoxifen and toremifene, their relatively long lifetimes,
Oxidation of 4-Hydr oxytam oxifen an d 4-Hydr oxy-
tor em ifen e by Cytoch r om e P 450. Oxidation of 4-hy-
droxytamoxifen to its quinone methide by cytochrome
P450 has been proposed as one of the tamoxifen bioac-
tivation pathways (20-22). P450 3A4 has been shown
to oxidize tamoxifen and its analogues to their corre-
sponding phase I metabolites in humans (50). In this
study, we used microsomes isolated from rats treated
with dexamethasone to induce P450 3A isozymes. In the
absence of GSH, we were able to detect the presence of
the 4-hydroxytamoxifen quinone methide generated from
microsomal incubations. It had the same retention time
and mass spectra as the standard 4-hydroxytamoxifen
quinone methide. This was surprising since several
studies in the past have indicated that most quinone
methides dimerize or disproportionate, undergo hydroly-
sis, or alkylate microsomal proteins in the absence of a
trapping agent (37, 38, 51-53) which suggests that direct
detection of these reactive intermediates would be dif-
ficult. These experiments further emphasize the unusual
stability of the 4-hydroxytamoxifen quinone methide
under physiological conditions. In terms of quantitative
work with radiolabeled GSH, after incubation and sepa-
ration by HPLC, the radiochromatograms gave peaks
with retention times and UV spectra identical to those
derived from addition of GSH to the synthetic quinone
methides (Table 2). Very low levels of GSH conjugates
were detected in microsomal incubations with either
4-hydroxytamoxifen or 4-hydroxytoremifene perhaps due
to the fact that little quinone methide was formed from
these phenols. 4-Hydroxytoremifene was found to be a
better substrate for quinone methide formation, generat-
ing approximately 4-fold more GSH trapped products
than 4-hydroxytamoxifen.
In the 4-hydroxytamoxifen incubations, three other
GSH-dependent peaks were detected, which were later
identified as GSH conjugates of 3,4-dihydroxytamoxifen-
o-quinone (Table 2) (54). This is not surprising since

Quinone Methides of Tamoxifen and Toremifene
Chem. Res. Toxicol., Vol. 13, No. 1, 2000 51
66, pp 253-365, International Agency for Research on Cancer,
Lyon, France.
and the instability of the tamoxifen-GSH conjugates
may indicate that the quinone methide pathway may
play a lesser role in the carcinogenic mechanism of
tamoxifen and toremifene. These conclusions are sup-
ported by recent reports which have suggested that
quinone methide formation from tamoxifen may not be
a major pathway in the formation of tamoxifen-derived
DNA adducts in rat models (20, 21). Instead, R-hydroxy-
lation of tamoxifen, sulfate ester formation, followed by
generation of the tamoxifen carbocation may represent
the principle carcinogenic pathway (15-17). Alterna-
tively, we have shown that 4-hydroxytamoxifen can be
converted to the 3,4-dihydroxytamoxifen-o-quinone which
could represent another potential cytotoxic pathway
which has received little attention (18, 19). This possibil-
ity is explored in ref 54.
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Duffel, M. W. (1998) R-Hydroxytamoxifen is
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Ack n ow led gm en t. We acknowledge Dr. Richard B.
van Breemen, Dr. Dejan Nikolic, and Mr. Lixin Shen for
assistance with mass spectrometry experiments and
Hewlett-Packard for providing the LC/MS instrumenta-
tion. We also thank Dr. Emily Pisha for assistance with
the cell culture experiments. This work was supported
by NIH Grant CA79870.
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