Identification of tamoxifen-DNA adducts induced by α-acetoxy-N-desmethyltamoxifen

Article abstract of DOI:10.1021/tx000074o

Treatment with tamoxifen increased the risk of endometrial cancers in breast cancer patients and women participating in the chemoprevention study. In our laboratory, tamoxifen-DNA adducts, including α-(N²-deoxyguanosinyl)tamoxifen (dG-N²-TAM), were detected in the endometrium of women taking tamoxifen [Shibutani, S., et al. (1999) Chem. Res. Toxicol. 12, 646-653]. On the basis of recent animal studies, deoxyguanosinyl-N-desmethyltamoxifen (dG-N-desmethylTAM) adducts are also suspected to be formed in the liver. In the study presented here, we synthesized α-acetoxy-N-desmethyltamoxifen as a model activated metabolite of N-desmethyltamoxifen. The overall yield of α-acetoxy-N-desmethyltamoxifen from α-hydroxy-tamoxifen was approximately 42%. α-Acetoxy-N-desmethyltamoxifen was highly reactive to 2'-deoxyguanosine, as was similarly observed for tamoxifen α-sulfate. The two reaction products were identified as a mixture of epimers of the trans form or cis form of α-(N²-deoxyguanosinyl)-N-desmethyltamoxifen (dG-N²-N-desmethylTAM) by mass and proton magnetic resonance spectroscopy. In addition, the trans and cis forms of dG 3'-monophosphate-N²-N-desmethylTAM were prepared as standard markers for ³²P-postlabeling/HPLC analysis. Using this technique, dG-N²-N-desmethylTAM adducts were detected in calf thymus DNA reacted with α-acetoxy-N-desmethyltamoxifen.

Full text of DOI:10.1021/tx000074o

Chem. Res. Toxicol. 2000, 13, 761-769
761
Id en tifica tion of Ta m oxifen -DNA Ad d u cts In d u ced by
r-Acetoxy-N-d esm eth ylta m oxifen
Masayuki Kitagawa,† Anisetti Ravindernath,† Naomi Suzuki,† Robert Rieger,†
Isamu Terashima,† Atsushi Umemoto,‡ and Shinya Shibutani*,†
Laboratory of Chemical Biology, Department of Pharmacological Sciences, State University of New
York at Stony Brook, Stony Brook, New York 11794-8651, and Second Department of Surgery,
School of Medicine, University of Tokushima, 3-18-15, Kuramoto-cho, Tokushima 770-8503, J apan
Received March 31, 2000
Treatment with tamoxifen increased the risk of endometrial cancers in breast cancer patients
and women participating in the chemoprevention study. In our laboratory, tamoxifen-DNA
adducts, including R-(N2-deoxyguanosinyl)tamoxifen (dG-N2-TAM), were detected in the
endometrium of women taking tamoxifen [Shibutani, S., et al. (1999) Chem. Res. Toxicol. 12,
646-653]. On the basis of recent animal studies, deoxyguanosinyl-N-desmethyltamoxifen (dG-
N-desmethylTAM) adducts are also suspected to be formed in the liver. In the study presented
here, we synthesized R-acetoxy-N-desmethyltamoxifen as a model activated metabolite of
N-desmethyltamoxifen. The overall yield of R-acetoxy-N-desmethyltamoxifen from R-hydroxy-
tamoxifen was approximately 42%. R-Acetoxy-N-desmethyltamoxifen was highly reactive to
2′-deoxyguanosine, as was similarly observed for tamoxifen R-sulfate. The two reaction products
were identified as a mixture of epimers of the trans form or cis form of R-(N2-deoxyguanosinyl)-
N-desmethyltamoxifen (dG-N2-N-desmethylTAM) by mass and proton magnetic resonance
spectroscopy. In addition, the trans and cis forms of dG 3′-monophosphate-N2-N-desmethylTAM
were prepared as standard markers for 32P-postlabeling/HPLC analysis. Using this technique,
dG-N2-N-desmethylTAM adducts were detected in calf thymus DNA reacted with R-acetoxy-
N-desmethyltamoxifen.
mechanisms, R-hydroxylation of these products (Figure
1), followed by sulfation or acetylation, may be a major
pathway capable of forming DNA adducts (16-20). Two
trans (fr-1 and fr-2) and two cis (fr-3 and fr-4) epimers
of R-(N2-deoxyguanosinyl)tamoxifen (dG-N2-TAM) DNA
adducts (12, 21, 22) can be produced via sulfation of
R-OHTAM by hydroxysteroid sulfotransferases (23, 24).
dG-N2-TAM adducts were miscoding and mutagenic
lesions, primarily generating G f T transversions in
mammalian cells (25, 26). These adducts are likely to be
the major DNA adducts in the liver of rats treated with
TAM or R-OHTAM (12, 27). dG-N2-TAM adducts (1.5-
13 adducts/108 nucleotides) were also detected in en-
dometrial tissues of women taking TAM (28, 29), con-
tradicting earlier results showing that such TAM adducts
were not significantly detected in other laboratories (30-
32).
In tr od u ction
The Breast Cancer Prevention Trial initiated by the
National Surgical Adjuvant Breast and Bowel Project
recently reported that tamoxifen (TAM),1 a chemothera-
peutic agent for breast cancer, reduces the risk of
development of breast cancer by 44-55% for healthy
women at high risk (1). However, treatment with TAM
increases the incidence of endometrial cancer in healthy
women enrolled in the NSABP chemopreventive trial (1)
as well as in breast cancer patients (2-4). The genotoxic
effects in the human uterus (5, 6) are suspected because
of the observation that TAM-induced hepatocellular
tumors in rats (7-9) were associated with the formation
of covalent DNA adducts (10-12) by the activated TAM
metabolites (13-15).
TAM is metabolically converted to R-hydroxytamoxifen
(R-OHTAM), N-desmethyltamoxifen (N-desmethylTAM),
tamoxifen N-oxide (TAM N-oxide), and 4-hydroxytamox-
ifen (4-OHTAM) (16-20) (the structures in Figure 1).
These compounds are precursors to reactive species
capable of damaging DNA. Among several proposed
Using mass spectroscopy, dG-N-desmethylTAM was
suggested to be another major adduct, in addition to dG-
N2-TAM, in the liver of rats treated with TAM (33). When
rats were treated with N-desmethylTAM, one major
adduct was also detected in the liver by a 32P-post-
labeling/HPLC analysis (34, 35). Thus, another major
adduct could be formed via sulfation or acetylation of
R-OH-N-desmethylTAM.
Recently, R-OH-N-desmethylTAM was synthesized via
desmethylation of 1-bromo-2-{4-[2-(dimethylamino)ethyl]-
phenyl}-1,2-diphenylethene (36). R-Sulfoxy-N-desmethyl-
TAM prepared from R-hydroxy-N-desmethylTAM was
shown to be reactive to DNA in vitro, identifying a trans
form of R-(deoxyguanosin-N2-yl)-N-desmethyltamoxifen
* To whom correspondence should be addressed. Phone: (631) 444-
8018. Fax: (631) 444-3218. E-mail: shinya@pharm.sunysb.edu.
† State University of New York at Stony Brook.
‡ University of Tokushima.
1
Abbreviations: dG, 2′-deoxyguanosine; dG3′P, 2′-deoxyguanosine
3′-monophosphate; TAM, tamoxifen; R-OHTAM, R-hydroxytamoxifen;
dG-N2-TAM, R-(N2-deoxyguanosinyl)tamoxifen; TAM N-oxide, tamox-
ifen N-oxide; R-OHTAM N-oxide, R-hydroxytamoxifen N-oxide; dG-N2-
TAM N-oxide, R-(N2-deoxyguanosinyl)tamoxifen N-oxide; N-des-
methyTAM, N-desmethyltamoxfen; dG-N2-N-desmethylTAM, R-(N2-
deoxyguanosinyl)-N-desmethyltamoxifen.
10.1021/tx000074o CCC: $19.00 © 2000 American Chemical Society
Published on Web 07/19/2000

762 Chem. Res. Toxicol., Vol. 13, No. 8, 2000
Kitagawa et al.
F igu r e 1. Metabolic pathway of tamoxifen.
(dG-N2-N-desmethylTAM) (36). However, the synthetic
step from 1-bromo-2-{4-[2-(methylamino)ethyl]phenyl}-
1,2-diphenylethene to R-OH-N-desmethylTAM was criti-
cal, and the yield was only 12%. In addition, the cis form
of the dG-N2-N-desmethylTAM adduct has not yet been
characterized.
In this paper, R-acetoxy-N-desmethylTAM was pre-
pared from R-OHTAM as a model activated form of
N-desmethylTAM (Scheme 1). The overall yield was
approximately 42%. R-Acetoxy-N-desmethylTAM was
highly reactive to dG, resulting in two reaction products
identified as a mixture of two epimers of the trans or cis
form of dG-N2-N-desmethylTAM. These trans and cis
isomers of dG 3′-monophosphate-N2-N-desmethylTAM
(dG3′P-N2-N-desmethylTAM) were also prepared and used
as standard markers for quantification of N-desmethyl-
TAM-DNA adducts by 32P-postlabeling/HPLC analysis.
(St. Louis, MO) and nuclease P1 and alkaline phosphatase (type
III) from Boehringer Mannheim (Indianapolis, IN). RNase A,
RNase T1, micrococcal nuclease, and spleen phosphodiesterase
were obtained from Worthington Biochemical Co. (Freehold,
NJ ). T4 polynucleotide kinase was purchased from Stratagene
(La J olla, CA).
Syn th esis of Ch em ica l Ma ter ia ls. A trans form of R-hy-
droxytamoxifen (R-OHTAM) was synthesized by the established
protocol (37). TAM R-sulfate and R-acetoxyTAM were synthe-
sized by the established method (12, 21).
Syn th esis of r-Acetoxy-N-d esm eth ylTAM. (E)-4-{4-[2-(N-
E t h oxyca r b on yl-N-m et h yla m in o)et h oxy]p h en yl}-3,4-d i-
p h en yl-3-bu ten -2-ol (2). To a solution of R-OHTAM (1, 99 mg,
0.26 mmol) in benzene (5 mL) was added ethylchloroformate
(0.05 mL), and the mixture was refluxed for 1 h. After the
solvent was removed by evaporation, the residue was chromato-
graphed on a column of silica gel with (a) 1:3 and (b) 1:2 ethyl
acetate/hexane. Eluant b gave 2 (87 mg, 0.20 mmol) as a syrup.
The yield of 2 was 79%. 1H NMR for compound 2 (CDCl3): δ
1.15-1.29 (6H, m), 2.95 (3H, s), 3.52 (2H, m), 3.94 (2H, m), 4.05
(2H, q, J ) 7.1 Hz), 4.83 (1H, q, J ) 6.5 Hz), 6.52 (2H, m), 6.81
(2H, m), 7.15-7.42 (10H, m).
Ma ter ia ls a n d Meth od s
Ca u tion : TAM R-sulfate and R-acetoxy-N-desmethylTAM are
potentially genotoxic and should be handled with proper care.
Ch em ica ls. Organic chemicals used for synthesis were
supplied by Aldrich Chemical (Milwaukee, WI) and Fisher
Scientific (Pittsburgh, PA). [γ-32P]ATP (specific activity of 6000
Ci/mmol) was obtained from Amersham Corp. (Arlington Heights,
IL). Polyethyleneimine (PEI)-cellulose plates were purchased
from Machery-Nagel (Duren, Germany). Calf thymus DNA,
proteinase K, and potato apyrase were purchased from Sigma
(E )-4-{4-[2-(N -Be n zyloxyca r b on yl-N -m e t h yla m in o)-
eth oxy]p h en yl}-3,4-d ip h en yl-3-bu ten -2-ol (4). A mixture of
2 (87 mg, 0.20 mmol), ethylene glycol (3 mL), hydrazine hydrate
(0.3 mL), and potassium hydroxide (500 mg) was stirred at 140
°C for 2 h. The reaction mixture was poured into ice/water and
extracted with diisopropyl ether. The extract was washed with
brine and dried using anhydrous sodium sulfate, and then the
solvent was evaporated. The residual syrup was dissolved in
methanol (5 mL) (added at 0 °C), triethylamine (109 µL), and

dG-N2-N-Desmethyltamoxifen-DNA Adducts
Chem. Res. Toxicol., Vol. 13, No. 8, 2000 763
benzylchloroformate (56 µL). The mixture was stirred at 0 °C
for 1 h, and then concentrated. The residue was extracted with
ethyl acetate, washed with brine, and evaporated. The residue
was chromatographed on a column of silica gel with toluene/
ethyl acetate (3:1) to give 4 (95 mg, 0.19 mmol) as a syrup. The
desmethylTAM-modified dG3′P, the pooled butanol extracts were
concentrated at reduced pressure and subjected to HPLC, as
similarly described for isolation of dG-N2-N-desmethylTAM. A
part of the purified N-desmethylTAM-modified dG3′P was evapo-
rated to dryness and incubated at 37 °C for 1 h with alkaline
phosphatase (3 units) in 100 µL of 100 mM Tris-HCl buffer (pH
7.0). The reaction mixture was extracted twice with 100 µL of
butanol. The butanol extract was evaporated at reduced pres-
sure and subjected to a reverse-phase column using the same
HPLC condition described for dG-N2-N-desmethylTAM. The
retention time and UV spectra of the products were compared
with that of diastereoisomers of dG-N2-N-desmethylTAM. The
molecular weight of N-desmethylTAM-modified dG3′P was also
measured by LC/mass spectrometry.
Rea ction of DNA w ith Activa ted F or m s of TAM An a -
logu es. Aliquots of DNA (10 µg) were incubated at 37 °C for 1
h with 400 µg of TAM R-sulfate or R-acetoxy-N-desmethylTAM
in 100 µL of 100 mM Tris-HCl buffer (pH 8.0). The samples were
evaporated to dryness in a vacuum desiccator, and extracted
three times with 1.0 mL of ethanol. The recovered DNA (0.4
µg) was digested at 37 °C for 2 h in 30 µL of 17 mM sodium
succinate buffer (pH 6.0) containing 8 mM CaCl2, using 1.5 units
of micrococcal nuclease and 0.15 unit of spleen phosphodi-
esterase. Afterward, 1.0 unit of nuclease P1 was added and the
reaction mixture was incubated at 37 °C for 1 h. Samples were
dissolved in 100 µL of distilled water and extracted twice with
200 µL of butanol. The butanol fraction was back-extracted with
50 µL of distilled water, dried, and then used for analysis of
TAM-DNA adducts. Approximately 95% of the TAM adducts
were recovered by butanol extraction. The concentration of the
DNA was estimated to be 50 µg per 1 OD260 unit.
1
yield of 4 from compound 2 was 93%. H NMR for compound 4
(CDCl3): δ 1.20 (3H, d, J ) 6.5 Hz), 2.98 (3H, s), 3.56 (2H, m),
3.95 (2H, m), 4.83 (1H, q, J ) 6.5 Hz), 5.10 (2H, s), 6.49 (2H,
m), 6.80 (2H, m), 7.12-7.43 (15H, m).
(E)-2-Acetoxy-4-{4-[2-(N-ben zyloxyca r bon yl-N-m eth yl-
a m in o)eth oxy]p h en yl}-3,4-d ip h en yl-3-bu ten e (5). To a so-
lution of 4 (95 mg, 0.19 mmol) in pyridine (4 mL) was added
acetic anhydride (1 mL), and the mixture was stirred at room
temperature for 15 h and then evaporated to a syrup. The
product was purified by chromatography on a column of silica
gel with toluene/ethyl acetate (4:1) to afford 5 (75 mg, 0.14
mmol) as a syrup. The yield of 5 from compound 4 was 73%. 1H
NMR for compound 5 (CDCl3): δ 1.27 (3H, d, J ) 6.6 Hz), 1.91
(3H, s), 2.98 (3H, s), 3.56 (2H, m), 3.95 (2H, m), 5.10 (2H, s),
5.76 (1H, q, J ) 6.6 Hz), 6.47 (2H, m), 6.78 (2H, m), 7.06-7.45
(15H, m). 1H NMR for compound 5 (CD3OD): δ 1.25 (3H, d, J
) 6.6 Hz), 1.88 (3H, s), 2.94 (3H, s), 3.56 (2H, t, J ) 5.5 Hz),
3.95 (2H, m), 5.05 (2H, brs), 5.74 (1H, q, J ) 6.6 Hz), 6.49 (2H,
m), 6.77 (2H, m), 7.10-7.44 (15H, m).
r-Acetoxy-N-d esm eth ylTAM (6) ((E)-2-Acetoxy-4-{4-[2-
(m eth yla m in o)eth oxy]p h en yl}-3,4-d ip h en yl-3-bu ten e). To
a solution of 5 (75 mg, 0.14 mmol) in ethyl acetate (10 mL) was
added 5% Pd-C catalyst (50 wt %, 10 mg), and the mixture was
stirred for 2 h at room temperature in a hydrogen atmosphere.
Filtration of the catalyst and evaporation of the solvent gave
R-acetoxy-N-desmethylTAM (6, 56 mg, 0.13 mmol) as a crude
syrup. The purity of 6 according to NMR data was ∼80%. The
yield of 6 from compound 5 was 79%. MS (FAB, positive): m/z
Qu a n tita tion of 32P -La beled DNA Ad d u cts by HP LC.
The digests of pooled extracts were incubated with 40 µCi of
[γ-32P]ATP (6000 Ci/mmol) and 3 µL of T4 polynucleotide kinase
(10 units/µL) (38) and developed for 16 h on a 10 cm × 10 cm
PEI-cellulose thin-layer plate using 1.7 M sodium phosphate
buffer (pH 6.0) with a paper wick. The position of adducts was
established by autoradiography, using Kodak Xomat XAR film.
32P-labeled products remaining on the TLC plate were recovered,
using 4 M pyridinium formate (pH 4.3), and evaporated to
dryness. The recovery of 32P-labeled products was approximately
84%. Using a minor modification of a method established by
Martin et al. (27), the 32P-labeled products were subjected to a
Hypersil BDS C18 analytical column (0.46 cm × 25 cm, 5 µm;
Shandon), eluted at a flow rate of 1.0 mL/min with an isocratic
condition of 2.0 M ammonium formate (pH 4.0) containing 20%
acetonitrile/methanol (6:1, v/v) over the course of 40 min
followed by a linear gradient from 20 to 45% over the course of
25 min. The radioactivity was monitored by a radioisotope
detector (Berthold LB506 C-1, ICON Scientific Inc.) lined to a
1
416 [M + H]+. H NMR for compound 6 (CD3OD): δ 1.25 (3H,
d, J ) 6.6 Hz), 1.88 (3H, s), 2.41 (3H, s), 2.88 (2H, t, J ) 5.0
Hz), 3.94 (2H, t, J ) 5.0 Hz), 5.74 (1H, q, J ) 6.6 Hz), 6.58 (2H,
m), 6.77 (2H, m), 6.95-7.43 (10H, m).
Rea ction of r-Acetoxy-N-d esm th ylTAM w ith d G. 2′-
Deoxyguanosine (70 mg) was reacted at 37 °C for 18 h with 56
mg of R-acetoxy-N-desmethylTAM (∼80% pure) in 35 mL of 100
mM Tris-HCl buffer (pH 8.0) and 1.75 mL of acetonitrile. The
reaction mixture was extracted twice with 30 mL of butanol,
and the pooled butanol extracts were evaporated to dryness. To
isolate N-desmethylTAM-modified dG, the pooled butanol ex-
tracts were subjected to a reverse-phase µBondapak C18 column
(0.78 cm × 30 cm, Waters), eluted over 45 min at a flow rate of
2.0 mL/min with a linear gradient of 0.05 M triethylammonium
acetate (pH 7.0) containing 10 to 70% acetonitrile (21, 23). An
isocratic HPLC condition consisting of 0.05 M triethylam-
monium acetate (pH 7.0, 65%) and acetonitrile (35%) was also
used for the purification. HPLC analysis was performed on a
Waters 990 HPLC instrument, equipped with a photodiode
array detector. Mass spectrometry was performed on a Platform
liquid chromatograph (Micromass). The dG-N-desmethylTAM
fraction was chromatographed using a HP1100 system (Hewlett-
Packard, Palo Alto, CA), equipped with a photodiode array
detector. A PLRP-S column (1.0 mm × 50 mm, 8 µm; Microm
Bioresources) was employed at a flow rate of 50 µL/min with a
linear gradient of 0.05 M triethylammonium acetate (pH 7.0)
containing 5 to 50% acetonitrile over the course of 35 min and
subsequently 50 to 95% acetonitrile over the course of 15 min.
The effluent was directed to the source of the mass spectrometer
operated in the positive or negative ion mode. The mass rage of
m/z 250-900 was scanned in 2 s. NMR spectroscopy was
performed on a Bruker 600 MHz NMR instrument. 1H NMR
data of dG reaction products are described in Table 1.
Waters 990 HPLC instrument. Standard stereoisomers of dG3′p
-
N2-TAM (23) and dG3′p-N2-N-desmethylTAM were also labeled
with 32P (38).
The relative adduct levels were calculated according to Levay
et al. (39), using disintegrations per minute instead of counts
per minute [(total disintegrations per minute in adducts)/1.15
× 1011 dpm, assuming that 5 µg of DNA represented 1.52 × 104
pmol of dN3′P and the specific activity of the [γ-32P]ATP was 5.06
× 107 dpm/pmol]. The detection limit was approximately 2.5 ×
10-10 adduct. The specific activity of [γ-32P]ATP was corrected
by calculating the extent of decay.
Resu lts a n d Discu ssion
Syn th esis of r-Acetoxy-N-d esm eth ylTAM. To pre-
pare R-acetoxy-N-desmethylTAM, we initially attempted
to synthesize R-OH-N-desmthylTAM via desmethylation
of 1-bromo-2-{4-[2-(dimethylamino)ethyl]phenyl}-1,2-
diphenylethene using the modified procedures of Foster
et al. (37) and Olofson et al. (40). Although the reaction
was performed at -70 °C, the 2-methylaminoethoxy
Rea ction of r-Acetoxy-N-d esm eth ylTAM w ith d G 3′-
Mon op h osp h a te. 2′-Deoxyguanosine 3′-monophosphate (dG3′P
,
1.0 mg) was reacted at 37 °C for 18 h with 0.8 mg of R-acetoxy-
N-desmethylTAM (∼80% pure) in 500 µL of 100 mM Tris-HCl
buffer (pH 8.0) and 25 µL of acetonitrile. The reaction mixture
was extracted twice with 500 µL of butanol. To isolate N-

764 Chem. Res. Toxicol., Vol. 13, No. 8, 2000
Sch em e 1. Syn th esis of r-Acetoxy-N-d esm eth ylta m oxifen
Kitagawa et al.
Ta ble 1. 1H NMR Sp ectr u m of r-(Deoxygu a n osin -N2-yl)-N-d esm eth ylta m oxifen Ad d u cts
δ (ppm)a,b
fr-1
fr-2
tamoxifen fragment
CH3CH
CH3NH
1.26, d, J ) 6.8 Hz
1.85,c bs
1.28, d, J ) 6.0 Hz
1.88,c bs
CH3NH
2.26, s
2.34, s
CH2NH
CH2O
CH3CH
2.70, t, J ) 5.6 Hz
3.82, t, J ) 5.6 Hz
4.90, m
2.83, t, J ) 5.6 Hz
4.01, t, J ) 5.6 Hz
4.95, m
H3 and H5
H2 and H6
phenyl
6.52, 6.54, each bd, J ) 8.8 Hz
6.76, 6.78, each bd, J ) 8.8 Hz
7.01-7.49, m
7.16, 7.18, each bd, J ) 8.4 Hz
7.33, 7.35, each bd, J ) 8.4 Hz
6.88-7.12, m
guanine fragment
N2H
5.96 and 6.01,c two d, J ) 7.4 Hz
5.95 and 6.01,c two d, J ) 7.4 Hz
H8
N1
7.97, s
7.98, s
10.08,c bs
10.08,c bs
sugar fragment
2′
5′
2.4-2.5, m
3.51, q
2.4-2.6, m
3.50, q
4′
3.88, m
3.90, m
3′
4.39, m
4.40, m
5′-OH
3′-OH
1′
4.78,c t, J ) 5.2 Hz
5.42,c d, J ) 3.3 Hz
6.18 and 6.22, two t, J ) 6.2 Hz
4.75,c t, J ) 5.2 Hz
5.34,c d, J ) 3.3 Hz
6.22 and 6.26, two t, J ) 6.2 Hz
a
b
Recorded in DMSO-d6. Abbreviations: s, singlet; d, doublet; t, triplet; q, quartet; m, multiplet; b, broad. c Can be exchanged with
D2O.
moiety was readily cleaved from the brominated com-
pounds. Using a similar synthetic method at a temper-
ature of less than -100 °C, Gamboa da Costa et al. (36)
synthesized R-OH-N-desmethylTAM. The lower reaction
temperature may be required for synthesis of such an
unstable compound. However, the yield of R-OH-N-
desmethylTAM was only 12% (36). Therefore, we used
the alternative procedure to synthesize R-acetoxy-N-
desmethylTAM, as shown in Scheme 1. Treatment of
R-OHTAM with ethylchloroformate in benzene gave 2 in
79% yield. Benzyloxycarbonyl compound 4 was prepared
by the following two processes: hydrazine hydrate/KOH
and benzylchloroformate/MeOH. Compound 4 was con-
verted into the corresponding acetate 5 by acetic anhy-

dG-N2-N-Desmethyltamoxifen-DNA Adducts
Chem. Res. Toxicol., Vol. 13, No. 8, 2000 765
F igu r e 2. HPLC separation of dG-N-desmethyltamoxifen formed by R-acetoxy-N-desmethyltamoxifen. dG (70 mg) was reacted at
37 °C for 18 h with R-acetoxy-N-desmethylTAM (56 mg) in 100 mM Tris-HCl buffer (pH 8.0), as described in Materials and Methods.
The reaction mixture was extracted twice with butanol, and the pooled butanol extracts were evaporated to dryness. (A) To isolate
dG-N-desmethylTAM (fr-1 and fr-2), 9 mg of the pooled butanol extracts was subjected to a reverse-phase µBondapak C18 column
(0.78 cm × 30 cm), eluted over the course of 45 min at a flow rate of 2.0 mL/min with a linear gradient of 0.05 M triethylammonium
acetate (pH 7.0) containing 10 to 70% acetonitrile. (B) dG-N-desmethylTAM (fr-2) was further purified by an isocratic HPLC condition
using 0.05 M triethylammonium acetate (pH 7.0, 65%) and acetonitrile (35%).
drite/pyridine. Finally, hydrogenolysis of 5 in the pres-
ence of a H2/5%Pd-C/EtOAc mixture gave R-acetoxy-N-
desmethylTAM 6 as an unsuitable syrup. The overall
yield from R-OHTAM was approximately 42%. Since
R-acetoxy-N-desmethylTAM was highly unstable upon
exposure to the atmosphere as was observed for TAM
R-sulfate (21), the purity of R-acetoxy-N-desmethylTAM
was approximately 80%, which is based on the NMR
data. Thus, this synthetic procedure can be used to
prepare R-acetoxy-N-desmethylTAM with reasonable
yield.
Ch a r a ct er iza t ion of d G P r od u ct s In d u ced b y
r-Acetoxy-N-d esm eth ylTAM. dG was reacted with
R-acetoxy-N-desmethylTAM as described in Materials
and Methods. The reaction products were extracted with
butanol from the reaction mixture, and isolated by a
preparative HPLC column using a linear gradient of 0.05
M triethylammonium acetate containing 10 to 70%
acetonitrile (Figure 2A). The retention times of fr-1 and
fr-2 were 29.7 and 31.6 min, respectively. Since fr-2 was
contaminated with other compounds, this fraction was
further purified by the isocratic HPLC system using 0.05
M triethylammonium acetate and acetonitrile (65:35)
(Figure 2B). Fr-2 was isolated at 22.8 min. When fr-1 or
fr-2 was subjected to an analytical HPLC column (panels
A and C of Figure 3), both fractions have two peaks
exhibiting identical UV spectra (panels B and D of Figure
3). The UV maximum (251 nm) of fr-1 was slightly
different from that of fr-2 (247 nm). Using positive ion
LC/mass spectroscopy, the protonated molecular ion of
fr-1 and fr-2 exhibited a peak at m/z 623 (Figure 4),
showing the molecular weight to be 622 Da. Other peaks
in this spectrum were due to background from the
chromatographic system. Thus, these products are sus-
pected to be the diastereoisomers of dG-N-desmethyl-
TAM, as was similarly observed for dG-N2-TAM (21) and
dG-N2-TAM N-oxide (41).
TAM N-oxide (41), and by D2O exchange for analyzing
the exchangeable NH and OH protons (Table 1). Two
doublet signals corresponding to monosubstituted amino
group (N2H) were observed at δ 5.96 and 6.01 in fr-1 and
at δ 5.95 and 6.01 in fr-2. Two triplet signals correspond-
ing to sugar H1′ were detected at δ 6.18 and 6.22 in fr-1
and at δ 6.22 and 6.26 in fr-2. This indicates that both
fr-1 and fr-2 contain two epimers. The integration values
of these peaks authenticates the existence of two epimers
in a ratio of 1:1 for fr-1 and 3:2 for fr-2. These epimers
could be produced via a carbocation generated at the
R-position of the N-desmethylTAM moiety, as observed
for TAM R-sulfate (42). Since fr-1 contains two epimers,
the two doublet signals corresponding to protons H3 and
H5, and H2 and H6, of alkoxyphenyl were overlapped
and observed at δ 6.53 and 6.77, respectively (Figure 5).
This indicates that fr-1 is a mixture of two trans epimers
of dG-N2-N-desmethylTAM. The NMR spectrum of fr-1
was consistent with that observed recently by Gamboa
da Costa (36).
On the other hand, the two doublet signals in fr-2 were
shifted downfield (δ 7.17 and 7.34) (Figure 5), confirming
that fr-2 is a mixture of two cis epimers of dG-N2-N-
desmethylTAM (21, 37, 41). The doublets of N2H at δ 5.96
and 6.01 for fr-1 and at δ 5.95 and 6.01 for fr-2 were
coupled to the methine moiety at δ 4.90 and 4.95,
respectively. The shift in the signals for proton H8 of
guanine indicates that the TAM moiety is linked to the
N2 position of guanine (Table 1 and Figure 5). Thus, fr-1
and fr-2 were identified as the mixture of epimers of dG-
trans-N2-N-desmethylTAM and dG-cis-N2-N-desmethyl-
TAM, respectively.
Detection of N-Desm eth ylTAM-DNA Ad d u cts. To
prepare dG3′P-N2-N-desmethylTAM that is used as a
standard for 32P-postlabeling analysis, dG3′P was reacted
with R-acetoxy-N-desmethylTAM. As shown in Figure
6A, three fractions (a , tR ) 20.3 min; b, tR ) 21.5 min;
and c, tR ) 26.6 min) were separated via HPLC. The UV
spectra of fractions a and b were similar to that of the
epimers of the trans form of dG-N2-N-desmethylTAM,
and the UV spectrum of fraction c was similar to that of
the epimers of the cis form of dG-N2-N-desmethylTAM
The structures of fr-1 and fr-2 were analyzed by 500
or 600 MHz 1H NMR. The spectra were recorded in
DMSO, and the assignment of the peaks was determined
by comparison with the NMR data of the trans and cis
forms of R-OHTAM (37), dG-N2-TAM (21), and dG-N2-

766 Chem. Res. Toxicol., Vol. 13, No. 8, 2000
Kitagawa et al.
F igu r e 3. HPLC pattern and UV spectra of dG-N2-N-desmethyltamoxifen. Partially purified fr-1 (A) and fr-2 (C) were subjected to
the analytical µBondapak C18 column (0.39 cm × 30 cm), eluted over the course of 45 min at a flow rate of 1.0 mL/min with a linear
gradient of 0.05 M triethylammonium acetate (pH 7.0) containing 10 to 70% acetonitrile. During the isolation of dG-N-desmethylTAM
by HPLC, UV spectra (230-400 nm) of fr-1 (B) and fr-2 (D) were monitored every 2 s using a multiple-photodiode array detector
(Waters).
F igu r e 4. Mass spectrum of dG-N2-N-desmethytamoxifen. Fr-
1, whose spectrum is shown in Figure 2A, was subjected to
positive ion FAB mass spectroscopy.
(Figure 6B). A part of each fraction was evaporated to
dryness, incubated with alkaline phosphatase to produce
the deoxynucleoside, and subjected to HPLC. The reten-
F igu r e 5. 1H NMR spectra of trans and cis forms of dG-N2-
N-desmethytamoxifen. Fr-1 and fr-2, whose spectr are shown
in panels A and B of Figure 2, were further purified using the
same HPLC system and identified by 500 or 600 MHz NMR, as
described in Table 1.
tion times of products obtained from fractions a and b
were consistent with fr-1-1 and fr-1-2, an epimer of the
trans form of dG-N2-N-desmethylTAM (data not shown).
Two products (fr-2-1 and fr-2-2) were observed in fraction
c; the retention times were also consistent with those of
the epimers of the cis form of dG-N2-N-desmethylTAM.
The ratio of fr-2-1 to fr-2-2 was 45:55. Using negative
ion LC/mass spectroscopy, the deprotonated molecular
ions of fractions a (Figure 7), b, and c (data not shown)
exhibited peaks at m/z 701, showing the molecular mass
to be 702 Da. Background ions at m/z 338 also appeared
in this spectrum. Thus, fractions a and b were the
epimers of the trans form of dG3′P-N2-N-desmethylTAM,
and fraction c was a mixture of epimers of the cis form
of dG3′P-N2-N-desmethylTAM.

dG-N2-N-Desmethyltamoxifen-DNA Adducts
Chem. Res. Toxicol., Vol. 13, No. 8, 2000 767
F igu r e 8. 32P-postlabeling/HPLC analysis of dG3′P-N2-N-des-
methytamoxifen. Standard stereoisomers of dG3′P-N2-N-des-
methylTAM (a -c) were labeled with 32P and developed for 16
h on a PEI-cellulose TLC plate using 1.7 M sodium phosphate
buffer (pH 6.0) as described in Materials and Methods. 32P-
labeled products remaining on the TLC plate were recovered
using 4 M pyridinium formate (pH 4.3) and evaporated to
dryness. 32P-labeled products were subjected to a Hypersil BDS
C18 analytical column (0.46 cm × 25 cm), eluted at a flow rate
of 1.0 mL/min with an isocratic condition of 2.0 M ammonium
formate (pH 4.0) containing 20% acetonitrile/methanol (6:1, v/v)
over the course of 40 min followed by a linear gradient from 20
to 45% over the course ofr 25 min. The radioactivity was
monitored by a radioisotope detector connected to a Waters 990
HPLC instrument.
were detected (Figure 9A). Fr-2 was detected as the major
adduct, and its retention time was consistent with that
of the peak b, an epimer of the trans form of dG3′P-N2-
N-desmethylTAM. Fr-1 and fr-3 were detected as the
minor adducts. The retention times of fr-1 and fr-3 were
consistent with those of peaks a and c, epimers of the
trans and cis forms, respectively, of dG3′P-N2-N-des-
methylTAM (Figure 9A). The levels of fr-1, fr-2, and fr-3
were 1.2, 8.7, and 0.9 adducts/105 nucleosides, respec-
tively. When the DNA was incubated only in the buffer,
no DNA adducts were detected (data not shown).
When TAM adducts in the livers of rats treated with
TAM or N-desmethylTAM were analyzed by a similar 32P-
postlabeling/HPLC technique, the retention time of the
second major adduct induced by TAM was consistent with
that of the major adduct observed with N-desmethylTAM
(34, 35). In fact, when DNA was reacted with R-acetoxy-
TAM, two epimers of the trans form of dG-N2-TAM
adducts (fr-1 and fr-2) and a mixture of two epimers of
the the cis form (fr-3) were detected at 23.8, 29.6, and
60.3 min, respectively (Figure 9B). Via cochromatography
of dG-N2-TAM adducts with dG-N2-N-desmethylTAM
adducts, the retention time of one epimer (fr-1) of the
trans form of dG-N2-TAM was consistent with one epimer
(fr-2) of the trans form of dG-N2-N-desmethylTAM (Fig-
ure 9C).
F igu r e 6. HPLC separation of dG3′P-N2-N-desmethyltamoxifen.
dG3′P (1.0 mg) was reacted at 37 °C for 18 h with R-acetoxy-N-
desmethylTAM (0.8 mg) in 100 mM Tris-HCl buffer (pH 8.0),
as described in Materials and Methods. The reaction mixture
was extracted twice with butanol. (A) To isolate N-desmethyl-
TAM-modified dG3′P, the pooled butanol extracts were concen-
trated at reduced pressure and subjected to HPLC, as similarly
described for isolation of dG-N2-N-desmethylTAM. (B) UV
spectra of the of trans (a and b) and cis forms (c) of dG3′P-N2-
N-desmethylTAM.
F igu r e 7. Mass spectrum of dG3′P-N2-N-desmethytamoxifen.
Fraction a , whose spectrum is shown in Figure 6A, was
subjected to negative ion FAB mass spectroscopy.
When these trans and cis forms of dG3′P-N2-N-des-
methylTAM were labeled with 32P, and analyzed by using
32P-postlabeling/HPLC on-line with a radioisotope detec-
tor, as described in Materials and Methods, the retention
times of a , b, and c were 20.9, 24.0, and 59.5 min,
respectively (Figure 8). When a calf thymus DNA was
reacted with R-acetoxy-N-desmethylTAM, three adducts
Using the same 32P-postlabeling/HPLC condition as in
the study presented here, 16 endometrial samples col-
lected from women taking TAM were analyzed (29). We
detected trans and cis epimers of dG-N2-TAM as the
major adducts in eight patients treated for various
periods of time with TAM. Fr-1, termed one of the trans

768 Chem. Res. Toxicol., Vol. 13, No. 8, 2000
Kitagawa et al.
adducts observed in tissues of animals treated with TAM
and in endometrial samples of women undergoing TAM
treatment will be identified.
Ack n ow led gm en t. This research was supported by
National Institute of Environmental Health Sciences
Grant ES09418.
Refer en ces
(1) Fischer, B., Costantino, J . P., Redmond, C. K., Fisher, E. R.,
Wickerham, D. L., and Cronin, W. M. (1994) Endometrial cancer
in tamoxifen-treated breast cancer patients: Findings from the
National Surgical Adjuvant Breast and Bowel Project (NSABP,
B-14). J . Natl. Cancer Inst. 86, 527-537.
(2) Clarke, M., Cillins, R., Davies, C., Godwin, J ., Gray, R., and Peto,
R. (1998) Tamoxifen for early breast cancer: An overview of the
randomized trials. Lancet 351, 1451-1467.
(3) Barakat, R. R. (1996) Endometrial cancer and tamoxifen. In
Tamoxifen: a Guide for Clinicians and Patients (J ordan, V. C.,
Ed.) pp 101-117, PRR Inc., Huntington, NY.
(4) American Cancer Society. Cancer facts and figures-1998 (1998)
American Cancer Society, Atlanta.
(5) Stearns, V., and Gelman, E. P. (1998) Does tamoxifen cause cancer
in human? J . Ciln. Oncol. 16, 779-792.
(6) Wogan, G. N. (1997) Review of the toxicology of tamoxifen. Semin.
Oncol. 24 (Suppl. 1), 87-97.
(7) Williams, G. M., Iatropoulos, M. J ., Djordjevic, M. V., and
Kaltenberg, O. P. (1993) The triphenylethylene drug tamoxifen
is a strong liver carcinogen in the rat. Carcinogenesis 14, 315-
317.
(8) Greaves, P., Goonetilleke, R., Nunn, G., Topham, J ., and Orton,
T. (1993) Two-year carcinogenicity study of tamoxifen in Alderley
Park Wistar-derived rats. Cancer Res. 53, 3919-3924.
(9) Hard, G. C., Iatropoulos, M. J ., J ordan, K., Radi, L., Kaltenberg,
O. P., Imondi, A. R., and Williams, G. M. (1993) Major difference
in the hepatocarcinogenicity and DNA adduct forming ability
between toremifene and tamoxifen in female Crl:CD(BR) rats.
Cancer Res. 53, 4534-4541.
(10) Han, X., and Liehr, J . G. (1992) Induction of covalent DNA
adducts in rodents by tamoxifen. Cancer Res. 52, 1360-1363.
(11) White, I. N. H., de Matteis, F., Davies, A., Smith, L. L., Crofton-
Sleigh, C., Venitt, S., Hewer, A., and Phillips, D. H. (1992)
Genotoxic potential of tamoxifen and analogues in female Fischer
F344/n rats, DBA/2 and C57B1/6 mice and in human MCL-5 cells.
Carcinogenesis 13, 2197-2203.
(12) Osborne, M. R., Hewer, A., Hardcastle, I. R., Carmichael, P. L.,
and Phillips, D. H. (1996) Identification of the major tamoxifen-
deoxyguanosine adduct formed in the liver DNA of rats treated
with tamoxifen. Cancer Res. 56, 66-71.
(13) Pathak, D. N., and Bodell, W. J . (1994) DNA adduct formation
by tamoxifen with rat and human liver microsomal activation
systems. Carcinogenesis 15, 529-532.
F igu r e 9. 32P-postlabeling/HPLC analysis of DNA reacted with
R-acetoxy-N-desmethyltamoxifen and R-acetoxytamoxifen. Ali-
quots of calf thymus DNA (10 µg) were incubated 37 °C for 1 h
with 50 µg of R-acetoxy-N-desmethylTAM (A) or 400 µg of
R-acetoxyTAM (B) in 100 mM Tris-HCl buffer (pH 8.0). The
recovered DNA (0.4 µg) was digested using the nuclease P1
enrichment procedure and labeled with 32P, as described in
Materials and Methods. (C) Spectrum for a sample containing
a half of that from panel A and one-third of that from panel B.
These samples were analyzed using the 32P-postlabeling/HPLC
method for analysis of TAM-DNA adducts, as shown in the
legend of Figure 8.
(14) Pathak, D. N., Pongracz, K., and Bodell, W. J . (1995) Microsomal
and peroxidase activation of 4-hydroxy-tamoxifen to form DNA
adducts: comparison with DNA adducts formed in Sprague-
Dawley rats treated with tamoxifen. Carcinogenesis 16, 11-15.
(15) Hemminki, K., Widlak, P., and Hou, S. M. (1995) DNA adducts
caused by tamoxifen and toremifene in human microsomal system
and lymphocytes in vitro. Carcinogenesis 16, 1661-1664.
(16) Potter, G. A., McCague, R., and J arman, M. (1994) A mechanism
hypothesis for DNA adduct formation by tamoxifen hepatic
oxidative metabolism. Carcinogenesis 15, 439-442.
(17) Phillips, D. H., Potter, G. A., Horton, M. N., Hewer, A., Crofton-
Sleigh, C., J arman, M., and Venitt, S. (1994) Reduced genotoxicity
of [D5-ethyl]-tamoxifen implicates R-hydroxylation of the ethyl
group as a major pathway of tamoxifen activation to a liver
carcinogen. Carcinogenesis 15, 1487-1492.
(18) Phillips, D. H., Carmichael, P. L., Hewer, A., Cole, K. J ., and Poon,
G. K. (1994) R-Hydroxytamoxifen, a metabolite of tamoxifen with
exceptionally high DNA-binding activity in rat hepatocytes.
Cancer Res. 54, 5518-5522.
(19) J arman, M., Poon, G. K., Rowlands, M. G., Grimshaw, R. M.,
Horton, M. N., Potter, G. A., and McCague, R. (1995) The
deuterium isotope effect for the R-hydroxylation of tamoxifen by
rat liver microsomes accounts for the reduced genotoxicity of [D5-
ethtyl]tamoxifen. Carcinogenesis 16, 683-688.
epimers of dG-N2-TAM, was detected in six women taking
TAM; the level of adduct was 0.5-6.0 adducts/108 deoxy-
nucleotides (29). Since the retention time of fr-1 of dG-
N2-TAM was similar to that of fr-2 of dG-N2-N-des-
methylTAM, fr-1 detected in endometrial samples may
contain the trans epimer of the dG-N2-N-desmethyl-
TAM-DNA adduct.
We have now prepared standards of dG3′P-N2-N-des-
methylTAM for 32P-postlabeling analysis, in addition to
dG3′P-N2-TAM (23) and dG3′P-N2-TAM N-oxide (41). When
an appropriate HPLC system that can resolve all the iso
forms of these standards is established, TAM-DNA
(20) Poon, G. K., Walter, B., Lonning, P. E., Horton, M. N., and
McCague, R. (1995) Identification of tamoxifen metabolites in

dG-N2-N-Desmethyltamoxifen-DNA Adducts
Chem. Res. Toxicol., Vol. 13, No. 8, 2000 769
human Hep G2 cell line, human liver homogenate, and patients
on long-term therapy for breast cancer. Drug Metab. Dispos. 23,
377-382.
A., and Phillips, D. H. (1999) Lack of evidence from HPLC 32P-
post-labeling from tamoxifen-DNA adducts in the human en-
dometrium. Carcinogenesis 20, 339-342.
(21) Dasaradhi, L., and Shibutani, S. (1997) Identification of tamox-
ifen-DNA adducts formed by R-sulfate tamoxifen and R-acetoxy-
tamoxifen. Chem. Res. Toxicol. 10, 189-196.
(33) Rajaniemi, H., Rasanen, I., Koivisto, P., Peltonen, K., and
Hemminki, K. (1999) Identification of the major tamoxifen-DNA
adducts in rats liver by mass spectroscopy. Carcinogenesis 20,
305-309.
(34) Phillips, D. H., Hewer, A., Horton, M. N., Cole, K. J ., Carmichael,
P. L., Davis, W., and Osborne, M. R. (1999) N-Demethylation
accompanies R-hydroxylation in the metabolite activation of
tamoxifen in rat liver cells. Carcinogenesis 20, 2003-2009.
(35) Brown, K., Heydon, R. T., J ukes, R., White, I. N. H., and Martin,
E. A. (1999) Further characterization of the DNA adducts formed
in rat liver after the administration of tamoxifen, N-desmethyl-
tamoxifen or N,N-didesmethyltamoxifen. Carcinogenesis 20, 2011-
2016.
(22) Osborne, M. R., Hardcastle, I. R., and Phillips, D. H. (1997) Minor
products of reaction of DNA with R-acetoxytamoxifen. Carcino-
genesis 18, 539-543.
(23) Shibutani, S., Dasaradhi, L., Terashima, I., Banoglu, E., and
Duffel, M. W. (1998) R-Hydroxytamoxifen is
a substrate of
hydroxysteroid (alcohol) sulfotransferase, resulting in tamoxifen
DNA adducts. Cancer Res. 58, 647-653.
(24) Shibutani, S., Shaw, P., Suzuki, N., Dasaradhi, L., Duffel, M. W.,
and Terashima, I. (1998) Sulfation of R-hydroxytamoxifen cata-
lyzed by human hydroxysteroid sulfotransferase results in tamox-
ifen DNA adducts. Carcinogenesis 19, 2007-2011.
(25) Shibutani, S., and Dasaradhi, L. (1997) Miscoding potential of
tamoxifen-derived DNA adducts: R-(N2-deoxyguanosinyl)tamox-
ifen. Biochemistry 36, 13010-13017.
(26) Terashima, I., Suzuki, N., and Shibutani, S. (1999) Mutagenic
potential of R-(N2-deoxyguanosinyl)tamoxifen lesions, the major
DNA adducts detected in endometrial tissues of patients treated
with tamoxifen. Cancer Res. 59, 2091-2095.
(36) Gamboa da Costa, G., Hamilton, L. P., Beland, F. A., and
Marques, M. M. (2000) Characterization of the major DNA adduct
formed by R-hydroxy-N-desmethyltamoxifen in vitro and in vivo.
Chem. Res. Toxicol. 13, 200-207.
(37) Foster, A. B., J arman, M., Leung, O.-T., McCague, R., Leclercq,
G., and Devleeschouwer, N. (1985) Hydroxy derivatives of tamox-
ifen. J . Med. Chem. 28, 1491-1497.
(27) Martin, E. A., Heydon, R. T., Brown, K., Brown, J . E. B., Lim, C.
K., White, I. N. H., and Smith, L. L. (1998) Evaluation of
tamoxifen and R-hydroxytamoxifen 32P-post-labelled DNA adducts
by the development of a novel automated on-line solid-phase
extraction HPLC method. Carcinogenesis 19, 1061-1069.
(28) Shibutani, S., Suzuki, N., Terashima, I., Sugarman, S. M.,
Grollman, A. P., and Pearl, L. M. (1999) Tamoxifen-DNA adducts
in the endometrium of women treated with tamoxifen. Chem. Res.
Toxicol. 12, 646-653.
(29) Shibutani, S., Ravindernath, A., Suzuki, N., Terashima, I.,
Sugarman, S. M., Grollman, A. P., and Pearl, L. M. (2000)
Identification of tamoxifen-DNA adducts in the endometrium of
women treated with tamoxifen. Carcinogenesis (in press).
(30) Carmichael, P. L., Ugwumadu, A. H. N., Neven, P., Hewer, A. J .,
Poon, G. K., and Phillips, D. H. (1996) Lack of genotoxicity of
tamoxifen in human endometrium. Cancer Res. 56, 1475-1479.
(31) Hemminki, K., Rajaniemi, H., Lindahl, B., and Moberger, B.
(1996) Tamoxifen-induced DNA adducts in endometrial samples
from breast cancer patients.Cancer Res. 56, 4374-4377.
(32) Carmichael, P. L., Sardar, S., Crooks, N., Neven, P., Van Hoof,
I., Ugwumadu, A., Bourne, T., Tomas, E., Hellberg, P., Hewer,
(38) Maniatis, T., Fritsch, E. F., and Sambrook, J . (1982) Molecular
Cloning. A Laboratory Manual, Cold Spring Harbor Laboratory
Press, Cold Spring Harbor, NY.
(39) Levay, G., Pongracz, K., and Bodell, W. J . (1991) Detection of DNA
adducts in HL-60 cells treated with hydroquinone and p-benzo-
quinone by 32P-postlabeling. Carcinogenesis 12, 1181-1186.
(40) Olofson, R. A., Martz, J . T., Senet, J .-P., Piteau, M., and Malfroot,
T. (1984) A new reagent for the selective, high-yield N-dealkyl-
ation of tertiary amines: improved syntheses of naltrexone and
nalbuphine. J . Org. Chem. 49, 2081-2082.
(41) Umemoto, A., Monden, Y., Komaki, K., Suwa, M., Kanno, Y.,
Suzuki, M., Lin, C., Ueyama, Y., Momen, M. A., Ravindernath,
A., and Shibutani, S. (1999) Tamoxifen-DNA adducts formed by
R-acetoxytamoxifen N-oxide. Chem. Res. Toxicol. 12, 1083-1089.
(42) Sanchez, C., Shibutani, S., Dasaradhi, L., Bolton, J . L., Fan, P.
W., and McClelland, R. A. (1998) Lifetime and reactivity of an
ultimate tamoxifen carcinogen: The tamoxifen carbocation. J . Am.
Chem. Soc. 120, 13513-13514.
TX000074O