Peroxidase-mediated dealkylation of tamoxifen, detected by electrospray ionization-mass spectrometry, and activation to form DNA adducts

Article abstract of DOI:10.1016/j.freeradbiomed.2011.10.433

Tamoxifen (TAM) is extensively used for the treatment and prevention of breast cancer. Associated with TAM treatment is a two- to eightfold increase in risk of endometrial cancer. To understand the mechanisms associated with this increased risk several pa

Full text of DOI:10.1016/j.freeradbiomed.2011.10.433

Free Radical Biology & Medicine 52 (2012) 340–347
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Free Radical Biology & Medicine
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Original Contribution
Peroxidase-mediated dealkylation of tamoxifen, detected by electrospray
ionization–mass spectrometry, and activation to form DNA adducts
a,
b
Nilesh W. Gaikwad ⁎, William J. Bodell
a
Department of Nutrition and Department of Environmental Toxicology, University of California at Davis, Davis, CA 95616, USA
Contrast Therapeutics, Richmond, CA 94806, USA
b
a r t i c l e i n f o
a b s t r a c t
Article history:
Tamoxifen (TAM) is extensively used for the treatment and prevention of breast cancer. Associated with TAM
treatment is a two- to eightfold increase in risk of endometrial cancer. To understand the mechanisms
associated with this increased risk several pathways for TAM metabolism and DNA adduct formation
have been studied. The purpose of this study was to investigate the role of peroxidase enzymes in the metab-
olism of TAM and its activation to form DNA adducts. Using advanced tandem mass spectrometry we have in-
vestigated the peroxidase-mediated metabolism of TAM. Incubation of TAM with horseradish peroxidase
Received 12 April 2011
Revised 9 October 2011
Accepted 11 October 2011
Available online 18 October 2011
Keywords:
Tamoxifen
Metabolite E
DNA adducts
Peroxidase
HRP
Quinone methide
UPLC
ESI–MS/MS
Free radicals
2 2
(HRP) and H O produced multiple metabolites. Electrospray ionization–MS/MS analysis of the metabolites
demonstrated a peak at 301.3 m/z with daughter ions at 183.0, 166.9, 128.9, and 120.9 m/z, which identified
the metabolite as metabolite E (ME). The levels of ME were significantly inhibited by the addition of ascorbic
acid to the incubation mixture. Co-incubation of either TAM or ME and DNA with HRP and H O produced
2
2
three DNA adducts with a RAL of 1.97± 0.01×10− and 8.45± 2.7×10 . Oxidation of ME with MnO
7
−7
pro-
duced metabolite E quinone methide (MEQM). Furthermore, incubation of either TAM or ME with HRP and
2
H
2
O
2
resulted in formation of MEQM. Reaction of calf thymus DNA with MEQM produced three DNA adducts
−7
with a RAL of 9.8± 1.0×10 . Rechromatography analyses indicated that DNA adducts 1, 2, and 3 formed in
the HRP activation of either TAM or ME were the same as those formed by the chemical reaction of DNA with
MEQM. The results of these studies demonstrate that peroxidase enzymes can both metabolize TAM to
form the primary metabolite ME and activate ME to a quinone methide intermediate, which reacts with
DNA to form adducts. It is possible that peroxidase enzymes or peroxidase-like activity in endometrium
could contribute to the formation of DNA damage and genotoxic effects in endometrium after TAM
administration.
Published by Elsevier Inc.
Tamoxifen is an antiestrogenic compound that has been
demonstrated to significantly reduce the rate and time to reoccurrence
of breast cancer [1–3]. More recently TAM and its derivatives have also
been used in the prevention of breast cancer [4–6]. However, associated
with these beneficial effects is an increased risk for endometrial cancer
in these patients [7–11]. These risks for endometrial cancer associated
with TAM therapy are very similar to the increased risks for breast
cancer associated with hormone replacement therapy [12,13]. In
both cases, long-term administration of an estrogen or estrogen-like
compound results in a small but significant increase risk for cancer
investigated. In rats and mice, the administration of TAM results in
dose-dependent increase in the formation of DNA adducts
a
[4,5,15,16], mutations [17–20], and liver tumors [21]. The formation
of TAM–DNA adducts has been detected in monkeys treated with
TAM [6,22], in human endometrial explants treated with TAM or its
metabolites [23,24], and in women administered TAM [25–30]. In
addition women administered TAM showed an increase in K-ras
mutations in endometrial tumor samples [31,32]. Although TAM–DNA
adducts in human samples have not been observed by all investigators
[25,26], these results suggest that TAM and its metabolites increase the
risk for endometrial cancer through genotoxic mechanisms.
[12–14].
The mechanisms by which TAM and other estrogen compounds
may increase the risk for cancer in women have been extensively
TAM is extensively metabolized to 4-hydroxytamoxifen (4-HO-TAM),
α-hydroxytamoxifen (α-HO-TAM), tamoxifen-N-oxide, desmethylta-
moxifen, metabolite Y, metabolite E, and others in humans as well as
animals [33–38]. These metabolites have been shown to accumulate
in endometrial tissues [39]. Our laboratory has been interested in the
mechanism by which peroxidase enzymes can both metabolize
compounds to form primary metabolites and oxidize these metabolites
to intermediates that react with DNA. In previous studies, we have
Abbreviations: HRP, horseradish peroxidase; TAM, tamoxifen; ME, metabolite E;
MEQM, metabolite E quinone methide; PEI, polyethyleneimine; RAL, relative adduct
level; UPLC, ultraperformance liquid chromatography; MS/MS, tandem mass spectrometry.
⁎
Corresponding author. Fax: +1 530 752 8966.
E-mail address: ngaikwad@ucdavis.edu (N.W. Gaikwad).
0891-5849/$ – see front matter. Published by Elsevier Inc.
doi:10.1016/j.freeradbiomed.2011.10.433

N.W. Gaikwad, W.J. Bodell / Free Radical Biology & Medicine 52 (2012) 340–347
341
demonstrated that two of TAM's metabolites, namely 4-HO-TAM and
ME, could be further oxidized by peroxidase enzymes to intermediates
that react with DNA to form adducts. These observations of TAM
metabolites have been complemented by the studies that have
demonstrated that catechol estrogens can be oxidized to their
corresponding quinones and that these quinones react with DNA
carrier solution to optimize MS parameters and the multiple reaction
monitoring (MRM) method for UPLC–MS/MS operation. The organic
extracts from the assay mixtures were infused at the source and
MS/MS spectra were recorded.
The most abundant transition/daughter ion with the lowest
accompanying background was selected for UPLC–MS/MS analysis.
UPLC–MS/MS analyses of the assay samples were conducted using
a Waters Acquity UPLC system connected with the Xevo-TQ triple-
quadrupole mass spectrometer. Analytical separations on the UPLC
system were conducted using an Acquity UPLC HSS T3 1.7-μm
column (1×150 mm) at a flow rate of 0.15 ml/min. The gradient
[
40,41].
The purpose of these studies has been to investigate the
peroxidase-dependent conversion of TAM to primary metabolites
and the further activation of these metabolites to intermediates that
can react with DNA. Peroxidase enzymes are of interest because of
their presence in extrahepatic tissues such as endometrium, which
is the site for increased cancer risk after TAM therapy [42–44]. Perox-
idase enzymes have been demonstrated to carry out N-demethylation
and N-oxidation reactions and possibly O-demethylation [45,46]. The
formation of N-desmethyltamoxifen and tamoxifen-N-oxide has been
shown in incubation mixtures containing TAM and HRP [47]. However,
the O-dealkylation of TAM by peroxidase enzymes to form ME has not
been studied.
started with 50% A (0.1% formic acid in H
2
O) and 50% B (0.1%
formic acid in CH CN), changed to 0% A over 5 min, followed by a
3
1-min linear gradient to 50% A, resulting in a total separation time
of 6 min. The elutions from the UPLC column were introduced into the
mass spectrometer and resulting data were analyzed and processed
using MassLynx 4.1 software.
HRP activation of TAM or ME to form DNA adducts
The products generated by peroxidase activation of phenols and
catechols are quinones and quinone methides. These quinones and
quinone methides can react with DNA via Michael addition reactions
to form DNA adducts [48,49]. The detection and quantitation of DNA
adducts formed by the metabolites of TAM have been studied by a
number of methods including 32P-postlabeling [50,51] and mass
spectrometry [52,53]. In addition to these methods, photoactivation
of TAM to form a fluorescent phenanthrene product provides a
novel method for the detection and comparison of TAM–DNA adducts
The incubation mixture consisted of 100 μM TAM or ME, 15 units
of HRP, 100 μM H
2 2
O , and 500 μg of purified calf thymus DNA in
10 mM K HPO , pH 7.4. The final volumes were 2 ml. The reactions
2
4
were stopped with the addition of 1 ml chloroform and extracted
with chloroform:isoamyl alcohol (24:1). After extraction, the DNA was
precipitated with 4 M sodium acetate and cold ethanol and dissolved
in 1 mM sodium chloride and 1.5 mM sodium acetate.
[54,60].
Reaction of DNA with MEQM
These studies have demonstrated for the first time the peroxidase-
mediated side-chain cleavage of TAM to yield ME and that ME can be
activated to MEQM, which reacts with DNA to form DNA adducts. The
DNA adducts formed by peroxidase activation of TAM and its metabolites
may be related to the unidentified DNA adducts observed in some of the
human samples.
2 2
MEQM was prepared using MnO [56,57]. MnO was activated by
heating at 60 °C for 12 h. Eight milligrams of ME was dissolved in 1 ml
of acetonitrile and cooled to 4 °C. One hundred milligrams of heat-
2
activated MnO was added to the solution and the reaction mixture
was stirred vigorously for 10 min. After filtration, an aliquot of the
product was used for recording the UV spectra or further reaction
with DNA. Approximately 2 mg of MEQM was incubated with 1 mg
Experimental procedures
2 4
of DNA in 2.0 ml of 10 mM K HPO , pH 7.4, for 2 h at 37 °C. After
ME was prepared and purified as described [55]. TAM, calf thymus
incubation, DNA was extracted and precipitated as described above.
2 2
DNA, HRP, ascorbic acid, and H O were purchased from Sigma Chemical
Co. (St. Louis, MO, USA). All other chemicals were of the highest analytical
grade available.
HPLC–fluorescence (FL) analysis of TAM adducts
From each sample, 1 mg of DNA was hydrolyzed to deoxyribonu-
cleosides with a mixture of 50 units of DNase I, 4 units of snake
venom phosphodiesterase, and 4.5 units of alkaline phosphatase in
Peroxidase metabolism of TAM
The incubation mixture consisted of 100 μM TAM, 15 units of HRP, and
2
0.5 ml of 50 mM Tris–HCl, 5 mM MgCl , pH 7.4, for 24 h at 37 °C.
1
00 μM H
were 2 ml. Control assays were conducted without the addition of either
or HRP. Inhibitor studies were conducted with the addition of
00 μM ascorbic acid to the assay mixture. The reactions were stopped
2
O
2
in 10 mM K
2
HPO
4
, pH 7.4. The volumes of the incubations
Protein was precipitated by inserting the reaction mixture into a
boiling water bath for 90 s and then immediately cooling in the ice.
The solution was filtered through Centricon microcentrifuge tubes
(Millipore, Billerica, MA, USA) and analyzed by HPLC–FL.
2 2
H O
5
by the addition of and extraction with 1 ml of chloroform. The organic
layers were concentrated, filtered, and used for HPLC (not shown),
MS/MS, and UPLC–MS/MS analysis.
HPLC–FL analysis of TAM adducts was carried out using a PerkinElmer
265 pump (PerkinElmer, Cupertino, CA, USA) coupled to a Econosphere
5 μ C18 (25×4.6 mm) column (Alltech, Deerfield, IL, USA). Postcolumn
photoactivation of the TAM derivatives was performed at 254 nm with
a knitted reactor coil (0.25 mm×5 m; Supelco, Bellefonte, PA, USA)
coupled to a PerkinElmer LC 40 fluorescence detector set at 250 and
400 nm for excitation and emission. The mobile phase for HPLC analysis
of TAM adducts consisted of 50 mM ammonium formate at a flow
rate of 1 ml/min, which was increased to 100% methanol over 50 min.
Fluorescence response was digitized with a PE 970 interface and
analyzed using TurboChrom 4 software (PerkinElmer).
MS/MS and UPLC–MS/MS analysis of TAM metabolites
A Xevo-TQ triple-quadrupole mass spectrometer (Waters, Milford,
MA, USA) recorded MS and MS/MS spectra using electrospray ionization
in positive-ion mode, with capillary voltage of 3.0 kV, extractor cone
voltage of 3 V, and detector voltage of 500 V. Cone gas flow was set at
5
0 L/h and desolvation gas flow was maintained at 600 L/h. Source
temperature and desolvation temperature were set at 150 and
3
2
3
2
50 °C, respectively. The collision energy was varied between 20 and
9 V to optimize daughter ions. The acquisition range was 20–500 Da.
P-postlabeling analysis of adducts
Pure ME was introduced to the source at a flow rate of 10 μl/min by
using a methanol:water (1:1) and 0.1% formic acid mixture as the
The P
4 μg of DNA as previously described [50,58,59]. The radioactive mixtures
1
nuclease-enhanced 32P postlabeling was performed using

342
N.W. Gaikwad, W.J. Bodell / Free Radical Biology & Medicine 52 (2012) 340–347
were applied to 10×10-cm PEI-cellulose plates (T.J. Baker, Phillipsburg,
NJ, USA) and the DNA adducts were resolved in the following solvents:
mode to support the formation of ME. As shown in Fig. 2, both
synthetic ME and the assay mixture showed a peak (301.25>
D
4
0
, 1.0 M sodium phosphate buffer, pH 6.8; D
.8 M urea, pH 3.5; D , 0.72 M sodium phosphate, 0.45 M Tris, and 7.6 M
urea, pH 8.7; and D , 1.7 M sodium phosphate, pH 6. In the D and D
solvents, the plates were developed from the bottom to 3–5 cm onto a
paper wick. In the D and D solvents they were developed at a right
1
, 2 M lithium formate and
t
182.87) at R of ~4.9 min, confirming the dealkylation of TAM.
2
Other minor peaks at 3.25 and 5.15 min in the assay mixture
could be either impurities or interference of other TAM metabolic
products with similar ions (m/z) in their fragmentation pattern.
Control studies were performed without the addition of either
3
0
1
2
3
angle to the previous direction of development to 3–5 cm onto a
paper wick. The DNA adducts were located by autoradiography using
Kodak XAR-5 film and a DuPont Chronex Lightning Plus intensifying
screen. The areas corresponding to adducts were scraped into scintillation
vials containing 5 ml of scintillation cocktail (Safety Solve; Research
Products, Inc., Mount Prospect, IL, USA) and the radioactivity was
determined by scintillation counting. Areas adjacent to the radio-
active spots were also scraped and counted for the determination
of the background radioactivity. The relative adduct levels were
calculated as previously described [50,58,59].
2 2
HRP or H O to the incubation mixture. Under these conditions,
formation of ME was not observed (not shown). The addition of
500 μM ascorbic acid to the incubation inhibited the formation of
ME by 98% (not shown).
It is interesting to note that the assay mixtures containing ME or
2 2
TAM and HRP/H O showed a peak at 299.2 m/z, indicating the presence
of MEQM. Moreover, the daughter ion spectra of the peak at 299.2 m/z
clearly matched the fragmentation pattern of synthesized MEQM,
confirming the presence of MEQM (Fig. 3). A UV spectral analysis of
ME showed absorption maxima at 237 and 278 nm (Fig. 4A). Incubation
of ME with MnO
The fragmentation pattern (Fig. 3A) and change in UV spectrum after
oxidation by MnO are consistent with ME being converted to the
2
resulted in shifting the absorption maxima to 358 nm.
Rechromatography analysis of individual adducts
2
Individual adducts were eluted from PEI-cellulose with intermittent
shaking using 0.5 ml of 1.5 M triethylammonium bicarbonate (pH 7.5).
The radioactivity of the eluted material was counted and aliquots
containing≈500 cpm activity were evaporated to dryness and redis-
solved in 5 μl of water. These samples were applied to PEI-cellulose
sheets and developed in one dimension. The solvent systems used
for these rechromatographies are given in the legend to Fig. 6.
corresponding quinone methide MEQM in the assay mixtures. Further,
MEQM was incubated with purified calf thymus DNA and the DNA
adducts formed were analyzed. 32P-postlabeling analysis of the DNA
reacted with MEQM demonstrated the formation of two principal
adducts 1 and 3 and one minor adduct 2 (Fig. 4B). The RAL produced
−
7
by this reaction was 9.8± 1.0×10
and the distribution of adducts
was 1, 43.3%; 2, 5.3%; and 3, 51.4%.
In subsequent experiments, DNA was included in the incubation
mixture of TAM with HRP/H . The formation of DNA adducts by
Results
2 2
O
2 2
activation of TAM with HRP/H O was investigated using P1-
We have investigated the metabolism of TAM by HRP/H
very sensitive tandem mass spectrometry. Organic extracts of assay
mixtures containing TAM, HRP, and H clearly showed a peak at
01.2 m/z, which after fragmentation resulted in daughter ions at
83, 166.9, 128.9, 120.9, and 104.9 m/z (Fig. 1A). This fragmentation
2
O
2
using
enhanced 32P postlabeling. The results of this study demonstrated
that addition of DNA to the incubation mixture containing TAM and
O
2 2
2 2
HRP/H O produced three DNA adducts (Fig. 5A). The RAL produced
−
7
3
1
by this reaction was 1.19± 0.91×10 and the distribution of adducts
was 1, 63.5%; 2, 30.1%; and 3, 6.3%. In vitro activation of ME with HRP
pattern was matched with the fragmentation pattern of standard
ME (Fig. 1B). Further UPLC–MS/MS analysis was carried out in MRM
and H
2 2
O also produced three DNA adducts (Fig. 5B) with a relative
−
7
adduct level 8.45± 2.7×10 . The distribution of the DNA adducts
183.0
B
100
166.9
%
104.9
120.9
1
28.9
301.2
300
0
m/z
320
20
40
60
80
100
120
140
160
180
200
220
240
260
280
182.9
100
A
166.8
%
0
104.9
1
20.9
301.2
1
28.9
m/z
320
20
40
60
80
100
120
140
160
180
200
220
240
260
280
300
2 2
Fig. 1. MS/MS spectra of (A) standard metabolite E and (B) metabolite E formed by peroxidase-catalyzed O-dealkylation of TAM in the presence of H O . A plausible fragmentation
of metabolite E (301.2 M+1 ion) leading to major peaks is presented.

N.W. Gaikwad, W.J. Bodell / Free Radical Biology & Medicine 52 (2012) 340–347
F1:MRM of 12 channels, ES+
343
3
01.25 > 182.87
7.244e+003
4.89 min
100
B
%
0
min
1.00
2.00
3.00
4.00
5.00
6.00
F1:MRM of 12 channels, ES+
01.25 > 182.87
4
.90 min
3
100
A
3.043e+003
%
0
min
1.00
2.00
3.00
4.00
5.00
6.00
2 2
Fig. 2. UPLC–MS/MS chromatogram of (A) standard metabolite E and (B) metabolite E formed by peroxidase-catalyzed O-dealkylation of TAM in the presence of H O .
was 1, 60.2%; 2, 34.1%; and 3, 5.5%. In incubations containing DNA, plus
either TAM or ME but not HRP or H , no DNA adducts were detected
under these conditions for analysis (data not shown).
2 2
To further study the DNA adducts produced by HRP/H O activation
of TAM and ME we used a HPLC–FL method initially described by
Sharma and colleagues [54,60]. The advantage of this method is that it
2
O
2
221.1
1
00
%
0
C
9
1.0
299.2
2
04.9
1
06.8
269.9
m/z
320
2
0
0
0
40
40
40
60
60
60
80
80
80
100
120
120
120
140
140
140
160
160
160
180
180
180
200
220
240
240
240
260
280
300
220.9
1
00
%
0
B
A
204.9
9
0.9
2
99.2
1
06.9
2
69.9
m/z
320
2
100
200
220
260
280
300
2
20.9
1
00
%
0
2
05.1
9
1.0
299.2
107.0
2
70.0
m/z
320
2
100
200
220
260
280
300
Fig. 3. MS/MS spectra of (A) synthesized MEQM, (B) MEQM formed by peroxidase-catalyzed oxidation of metabolite E in the presence of H
2 2
O , and (C) MEQM formed in the assay
mixture containing peroxidase, TAM, and H . A plausible fragmentation of MEQM (299.2 M+1 ion) leading to major peaks is presented.
2 2
O

344
N.W. Gaikwad, W.J. Bodell / Free Radical Biology & Medicine 52 (2012) 340–347
3
2
1
0
ensures that a TAM-derived adduct is being quantified. HPLC–FL analysis
of DNA co-incubated with TAM and HRP/H showed the presence of a
single DNA adduct (a) with an R of 24.3 min (Fig. 5A). Analysis of DNA
also demonstrated the presence of
A
2 2
O
t
2 2
incubated with ME and HRP/H O
adduct a at 24.3 min (Fig. 5B).
Rechromatography experiments were performed to compare
adducts formed by HRP activation of TAM and ME with the adducts
formed by MEQM. The results of these comparisons demonstrated
that adduct 1 formed in each of the activation systems is the same.
Adduct 2 formed by HRP activation of either TAM or ME is the
same, and adduct 3 formed by HRP activation of TAM is the same as
adduct 3 formed by MEQM (Fig. 6).
358 nm
Discussion
300
400
500
The metabolism of TAM by a peroxidase enzyme has been investi-
gated. This study demonstrates that incubation of TAM with HRP/
Wavelength (nm)
2 2
H O resulted in O-dealkylation of TAM leading to the formation of
B
ME (Figs. 1 and 2). In addition to ME, several other products, which
have been previously reported, were detected [47] (not shown).
Furthermore, MS/MS study of the assay mixture containing TAM
and HRP/H
fragmentation patterns of the peak at 299.2 m/z were compared to
those formed in incubation mixtures of ME/HRP/H and ME/MnO
Fig. 3). The oxidation of ME with MnO was investigated by UV and
mass spectroscopy. Mixing of MnO with a solution of ME resulted in
2 2
O clearly suggested formation of MEQM when the
O
2 2
2
(
2
2
a shift of the UV spectrum maxima from 237 and 278 nm to 358 nm,
and the mass spectra of the incubation mixture clearly showed the
oxidation of ME to MEQM (Figs. 3A and 4A). Addition of ascorbic
acid to the incubation mixture reduced the level of ME as well as the
other products by 98% (not shown). This observation suggests a radical
intermediate in the formation of these products.
Incubation of DNA with MEQM resulted in the formation of two
principal (1 and 3) and one minor DNA adduct (2). Comparison of
the DNA adduct pattern produced by MEQM with that produced by
Fig. 4. (A) UV spectra of ME (dotted line) and MEQM (solid line) prepared by oxidation
3
2
of ME with MnO
2
. (B) P-postlabeling analysis of DNA adducts formed from incubation
of DNA with MEQM. The film was exposed for 8 h at −70 °C.
2 2
Fig. 5. Analysis of DNA adducts formed by the in vitro activation of (A) TAM or (B) ME with HRP and H O . The DNA adducts produced in these reactions were analyzed by HPLC–FL
3
2
(
left) and by P postlabeling (right). The films were exposed for 8 to 12 h at −70 °C.

N.W. Gaikwad, W.J. Bodell / Free Radical Biology & Medicine 52 (2012) 340–347
345
A
B
C
a
b
c
d
e
f
g
a
b
c
d
e
f
g
a
b
c
d
e
f
g
Fig. 6. Autoradiograms from rechromatography analysis of adducts 1, 2, and 3 formed by MnO
obtained from metabolite E activated (a) with MnO and (b) with HRP and from (c) tamoxifen and HRP. Lanes d and e are adduct 2 obtained from (d) metabolite E activated
with MnO and DNA and (e) tamoxifen and HRP. Lanes f and g are adduct 3 obtained from peroxidase activation of (f) metabolite E and (g) tamoxifen. (A) Lanes a–g were developed
in 2.48 M lithium hydroxide and 4.13 M urea, pH 3.35. (B) Lanes a–g were developed in 0.6 M lithium chloride, 0.37 M Tris, and 6.38 M urea, pH 8.0. (C) Lanes a–g were developed in
.3 M boric acid, 0.8 M NaCl, 6 mM EDTA, and 0.8 M urea, pH 8.0. The films were exposed for 50 h at −70 °C.
2
or HRP activation of ME and HRP activation of tamoxifen. Lanes a–c are adduct 1
2
2
0
ME/HRP/H
cases adduct 1 was a principal adduct. However, adduct 2 was the
other principal adduct from ME/HRP/H and adduct 3 from
MEQM. The change in adduct profiles of 2 and 3 produced after DNA
reaction with synthetic MEQM and ME/HRP/H could be attributed
to the distinct specific activation environment (Figs. 4B and 5B). These
results are consistent with ME being converted to MEQM, which reacts
with DNA to form the observed adducts.
2
O
2
indicates that they are similar but not identical. In both
to cytochrome P450, peroxidase enzymes can also metabolize TAM
to a number of primary metabolites. As demonstrated, of these primary
metabolites, ME can undergo further oxidation reactions to generate
reactive intermediates that bind to DNA. The results of these studies
suggest that peroxidase enzymes located at extrahepatic sites could
contribute to both the primary metabolism and the further oxidation
of TAM.
The formation of DNA adducts from the reaction of DNA with
quinones and quinone methides has been established [63–65]. In
these studies, the reaction of MEQM with DNA most probably occurs via
a 1,8 Michael addition reaction. A similar reaction has been demonstrated
to occur with the quinone methide formed by oxidation of 4-HO-TAM.
The site of reaction in DNA is probably the 2-amino of deoxyguanosine
(Fig. 7). Isolation and identification of the DNA adducts produced by
MEQM will be required to confirm this interpretation.
The mechanisms by which TAM and other estrogen compounds in-
crease the risk for cancer are currently unknown [65,66]. One possi-
bility is that these compounds are metabolized to intermediates that
react to form DNA adducts. Although the detection of TAM–DNA adducts
in endometrial samples has not been consistently observed [25–28], the
formation of depurinating DNA adducts by TAM metabolites may also be
possible [40,41]. The results of these studies suggest that peroxidase en-
zymes could contribute to this risk by metabolizing TAM to metabolites
that can be further activated to form DNA adducts.
2 2
O
2 2
O
Addition of DNA to the reaction mixture containing TAM and HRP/
2 2
H O P
resulted in the formation of DNA adducts as detected by both 32
postlabeling and HPLC–FL. Two principal adducts (1 and 2) and one
minor adduct (3) were detected by postlabeling analysis. When the
same samples were analyzed by HPLC–FL only a single DNA adduct
(
a) was detected. Activation of ME with HRP/H
adduct pattern that was the same as that observed with TAM and
HRP/H , with two major adducts and one minor adduct. In addition,
2 2
O resulted in a DNA
2
O
2
adduct a was also detected in this sample by HPLC–FL. The observed
differences in number of DNA adducts observed by postlabeling and
HPLC–FL suggest that under the HPLC conditions used adducts 1 and 2
are not resolved and are detected by HPLC–FL as a single adduct (a).
Based on postlabeling R
for adducts 1 and 2, it could be assumed that both these adducts will
have similar HPLC R ; however, there is a minor possibility that one of
the adducts could elute with the solvent front or a broad peak at
38 min. These results in conjunction with those of the metabolism
studies suggest that TAM was first converted to ME and then further
oxidized by HRP/H to form MEQM (Fig. 7).
f
values and perceived structural similarities
t
~
Acknowledgments
2 2
O
Earlier it was reported that, after its absorption, TAM is metabo-
lized to α-HO-TAM and 4-HO-TAM by cytochrome P450 3A4 in the
liver [61,62]. The results of this study demonstrate that in addition
This research was supported by a CHNR grant from the Department
of Nutrition, University of California at Davis, and by Grant CA 78237
from the National Cancer Institute.
Fig. 7. Proposed model for HRP activation of TAM to form ME and DNA adducts in vitro. This model illustrates how TAM undergoes O-dealkylation to give ME, which is further
activated by HRP to form the quinone methide MEQM. In this model MEQM undergoes a 1,8 Michael addition reaction with deoxyguanosine to form ME adduct.

346
N.W. Gaikwad, W.J. Bodell / Free Radical Biology & Medicine 52 (2012) 340–347
[
[
[
27] Shibutani, S.; Ravindernath, A.; Suzuki, N.; Terashima, I.; Sugarman, S. M.; Grollman,
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