Characterization of the major DNA adduct formed by α-hydroxy-N- desmethyltamoxifen in vitro and in vivo

Article abstract of DOI:10.1021/tx990187b

Tamoxifen is hepatocarcinogenic in rats and has been associated with an increased risk of endometrial cancer in women. Recent reports suggest that it may be genotoxic in humans. N-Desmethyltamoxifen is a major tamoxifen metabolite that has been proposed to be responsible for one of the major adducts detected in liver DNA of rats treated with tamoxifen. The metabolic activation of N-desmethyltamoxifen to DNA binding products may involve oxidation to α-hydroxy-N-desmethyltamoxifen followed by esterification. In the study presented here, we report the synthesis of α-hydroxy-N- desmethyltamoxifen and the characterization of the major adduct obtained from α-sulfoxy-N-desmethyltamoxifen in vitro as (E)-α-(deoxyguanosin-N²-yl)-N- desmethyltamoxifen. In addition, we use ³²P-postlabeling in combination with HPLC to compare the adducts formed in the livers of female Sprague- Dawley rats treated by gavage with tamoxifen or equimolar doses of α- hydroxy-N-desmethyltamoxifen. We conclude that one of the major adducts formed in vivo and previously suggested to derive from N-desmethyltamoxifen is chromatographically identical to α-(deoxyguanosin-N²-yl)-N- desmethyltamoxifen.

Full text of DOI:10.1021/tx990187b

200
Chem. Res. Toxicol. 2000, 13, 200-207
Ch a r a cter iza tion of th e Ma jor DNA Ad d u ct F or m ed by
r-Hyd r oxy-N-d esm eth ylta m oxifen in Vitr o a n d in Vivo
Gonc¸alo Gamboa da Costa,† L. Patrice Hamilton,‡ Frederick A. Beland,*,‡ and
M. Matilde Marques*,†
Centro de Qu´ımica Estrutural, Complexo I, Instituto Superior Te´cnico, Avenida Rovisco Pais,
1049-001 Lisboa, Portugal, and Division of Biochemical Toxicology, National Center
for Toxicological Research, J efferson, Arkansas 72079
Received November 8, 1999
Tamoxifen is hepatocarcinogenic in rats and has been associated with an increased risk of
endometrial cancer in women. Recent reports suggest that it may be genotoxic in humans.
N-Desmethyltamoxifen is a major tamoxifen metabolite that has been proposed to be responsible
for one of the major adducts detected in liver DNA of rats treated with tamoxifen. The metabolic
activation of N-desmethyltamoxifen to DNA binding products may involve oxidation to
R-hydroxy-N-desmethyltamoxifen followed by esterification. In the study presented here, we
report the synthesis of R-hydroxy-N-desmethyltamoxifen and the characterization of the major
adduct obtained from R-sulfoxy-N-desmethyltamoxifen in vitro as (E)-R-(deoxyguanosin-N2-
yl)-N-desmethyltamoxifen. In addition, we use 32P-postlabeling in combination with HPLC to
compare the adducts formed in the livers of female Sprague-Dawley rats treated by gavage
with tamoxifen or equimolar doses of R-hydroxy-N-desmethyltamoxifen. We conclude that
one of the major adducts formed in vivo and previously suggested to derive from N-desmethyl-
tamoxifen is chromatographically identical to R-(deoxyguanosin-N2-yl)-N-desmethyltamoxifen.
Tamoxifen has also been shown to induce a high fre-
quency of mutations in liver DNA of λ/lacI transgenic rats
(17). Furthermore, it has been associated with mutations
in the p53 tumor suppressor gene of liver tumors in rats
(18) and endometrial tumors in women (19). Recently,
DNA adducts have been reported in endometrial and
leucocyte samples from women undergoing tamoxifen
therapy (20-22); however, it has been suggested (23) that
the endometrial DNA adducts detected in one of these
studies (20) could arise from endogenous compounds. In
addition, other investigators (24, 25) have failed to detect
tamoxifen-DNA adducts in human endometrial samples.
A large body of evidence has indicated that one of the
metabolic pathways of tamoxifen activation to an elec-
trophilic derivative capable of binding to DNA involves
allylic oxidation to R-hydroxytamoxifen (2, Scheme 1,
pathway A), followed by esterification, most likely via
sulfation (26-32). In vitro reactions with synthetic model
esters of R-hydroxytamoxifen have led to the character-
ization of the major DNA adduct as (E)-R-(deoxyguanosin-
N2-yl)tamoxifen (3, Scheme 1), which exists as a mixture
of epimers at the allylic carbon. Minor adducts from
this reaction include the Z diastereomer of 3, and deoxy-
adenosine adducts, possibly at the exocyclic N6 through
the allylic carbon of tamoxifen (33-35). Using the 32P-
postlabeling methodology, adduct 3 has been identified
as one of the major adducts in the livers of rats treated
with tamoxifen on the basis of co-chromatography with
a synthetic standard (33, 36-38). Structural evidence for
the formation of 3 in rat liver in vivo has been presented
by Rajaniemi et al. (39) using mass spectrometry. Recent
work by Shibutani and co-workers (40) has shown
that all the diastereomers of R-(deoxyguanosin-N2-yl)-
tamoxifen are mutagenic and induce primarily G f T
transversions.
In tr od u ction
Tamoxifen1 (1, Scheme 1) has been used since the early
seventies as an adjuvant for the treatment of all stages
of hormone-dependent breast cancer (1, 2). Recent data
from a large-scale clinical trial in the United States have
indicated that it may also act as a chemoprotective agent
(3). Although this effect was not fully confirmed in a
British study (4, 5), tamoxifen has been approved by the
U.S. Food and Drug Administration for the prevention
of breast cancer in healthy women considered at high risk
for developing the disease. However, the prophylactic use
of this anti-estrogen is controversial because tamoxifen
treatment is associated with an increased risk of endo-
metrial cancer (1-3, 6, 7). In addition, the drug is
strongly hepatocarcinogenic in rats (8-10).
Although hormonal effects may account, in part, for
the carcinogenic properties of tamoxifen (11), the geno-
toxic potential of the drug has been confirmed by its
ability to form DNA adducts in rat liver (9, 12-16).
* To whom correspondence should be addressed. F.A.B.: HFT-110,
NCTR, J efferson, AR 72079; telephone, (870) 543-7205; fax, (870) 543-
7136; e-mail, fbeland@nctr.fda.gov. M.M.M.: CQE, Complexo I, IST,
Av. Rovisco Pais, 1049-001 Lisboa, Portugal; telephone, 351-21-841-
9200; fax, 351-21-846-4457; e-mail, qmdmar@beta.ist.utl.pt.
† Instituto Superior Te´cnico.
‡ National Center for Toxicological Research.
1 Abbreviations: Bis-Tris, bis(2-hydroxyethyl)iminotris(hydroxymeth-
yl)methane; Bu, n-butyl; Cquat, quaternary carbon; dG, 2′-deoxy-
guanosine; DMSO, dimethyl sulfoxide; dR, 2′-deoxyribosyl; EI, electron
impact; ESI, electrospray ionization; Gua, guanine; R-hydroxy-N-
desmethyltamoxifen, (E)-4-[4-[2-(methylamino)ethoxy]phenyl]-3,4-
diphenylbut-3-en-2-ol; R-hydroxytamoxifen, (E)-4-[4-[2-(dimethylamino)-
ethoxy]phenyl]-3,4-diphenylbut-3-en-2-ol; 4-hydroxytamoxifen, (Z)-1-
[4-[2-(dimethylamino)ethoxy]phenyl]-1-(4-hydroxyphenyl)-2-phenylbut-
1-ene; MS/MS, tandem mass spectrometry; PNK, T4 polynucleotide
kinase; tamoxifen, (Z)-1-[4-[2-(dimethylamino)ethoxy]phenyl]-1,2-
diphenylbut-1-ene.
10.1021/tx990187b CCC: $19.00 © 2000 American Chemical Society
Published on Web 02/26/2000

Major Adduct from R-Hydroxy-N-desmethyltamoxifen
Chem. Res. Toxicol., Vol. 13, No. 3, 2000 201
Sch em e 1. Meta bolic Activa tion P a th w a ys for Ta m oxifen a
a
Pathways A and C are the major activation routes in vivo. The adduct arising from pathway B has not been detected in vivo.
In addition to 3, a second major adduct and at least
methyltamoxifen have indicated that a metabolic path-
way via N-desmethyltamoxifen is a significant route to
DNA adducts in rat liver (37, 38), and structure 9 has
been assumed to correspond to one of the adducts.
However, no synthetic standards of N-demethylated
tamoxifen adducts have yet been available to test this
hypothesis.
In this paper, we report the synthesis of R-hydroxy-
N-desmethyltamoxifen and the characterization of the
major DNA adduct resulting from R-sulfoxy-N-desmeth-
yltamoxifen as (E)-R-(deoxyguanosin-N2-yl)-N-desmeth-
yltamoxifen. In addition, we compare DNA adduct for-
mation in female Sprague-Dawley rats treated by gavage
with tamoxifen and R-hydroxy-N-desmethyltamoxifen.
one additional minor adduct, which do not appear to be
derived from R-hydroxytamoxifen, are found in the livers
of tamoxifen-treated rats (36-38). Sequential oxidation
of tamoxifen to 4-hydroxytamoxifen (4, Scheme 1, path-
way B) and 4-hydroxytamoxifen quinone methide (5,
Scheme 1) or R,4-dihydroxytamoxifen (41) has been
proposed as a potential metabolic activation pathway of
tamoxifen that may lead to DNA binding products.
4-Hydroxytamoxifen is a major phase I metabolite of
tamoxifen in rat and human liver (6) and has been shown
to yield the 4-hydroxylated analogue of adduct 3 (6,
Scheme 1) upon oxidation and reaction with DNA in vitro
(42). Similarly, R,4-dihydroxytamoxifen yields DNA ad-
ducts when reacted with DNA at acidic pH (43). However,
subsequent studies in vivo (36, 44) have indicated that
metabolism via 4-hydroxytamoxifen is not a major acti-
vation pathway leading to DNA adducts in the rat.
The major phase I metabolite of tamoxifen in humans
(45-49) and rats (50, 51) is N-desmethyltamoxifen
(7, Scheme 1, pathway C), which can undergo further
metabolism at the allylic carbon (48, 52) to give R-
hydroxy-N-desmethyltamoxifen (8, Scheme 1). Subse-
quent conversion to a reactive ester may then lead to the
formation of DNA adducts. Rajaniemi et al. (39, 53)
presented the first evidence that one of the major
tamoxifen-related adducts in the livers of rats treated
with tamoxifen is N-demethylated. Comparisons between
the MS fragmentation patterns of this adduct and
R-(deoxyguanosin-N2-yl)tamoxifen (39) led to the sugges-
tion that the N-demethylated adduct is R-(deoxy-
guanosin-N2-yl)-N-desmethyltamoxifen (9, Scheme 1).
More recent data involving the administration of N-des-
Ma ter ia ls a n d Meth od s
Ca u tion : Tamoxifen and its derivatives are potentially geno-
toxic and should be handled with proper care. Exposure to 32P
should be kept as low as possible, by working in a confined
laboratory area, with protective clothing, plexiglass shielding,
Geiger counters, and body dosimeters. Waste materials must be
discarded according to appropriate safety procedures.
Ch em ica ls. Tamoxifen, salmon testes DNA, bis(2-hydroxy-
ethyl)iminotris(hydroxymethyl)methane (Bis-Tris), and enzymes
used in DNA hydrolysis were purchased from Sigma Chemical
Co. (St. Louis, MO). Carrier-free [γ-32P]ATP (>7000 Ci/mmol)
was purchased from ICN Pharmaceuticals (Costa Mesa, CA).
T4 polynucleotide kinase (PNK) was acquired from Amersham
U.S. Biochemical (Cleveland, OH). All other commercially
available reagents were obtained from Aldrich Chemical Co.
(Milwaukee, WI) or Sigma-Aldrich Qu´ımica, S.A. (Madrid,
Spain), and were used as received. Whenever necessary, solvents
were purified by standard procedures (54).

202 Chem. Res. Toxicol., Vol. 13, No. 3, 2000
Gamboa da Costa et al.
min, and the mixture was subsequently allowed to reflux
overnight. After the solvent was evaporated, the residue was
dissolved in methanol (10 mL) and refluxed for 45 min. The
solvent was then evaporated, and the mixture was redissolved
in methylene chloride and washed with a saturated sodium
bicarbonate solution. The organic layer was dried with anhy-
drous sodium sulfate, and the product was purified by flash
chromatography on silica gel H (type 60, E. Merck, Darmstadt,
Germany) by eluting with a stepwise gradient of 0 to 10%
methanol in methylene chloride. The N-demethylated product
11 was obtained in 98% yield as a 1/1 mixture of the E and Z
isomers, which were recrystallized from ethanolic hydrogen
chloride as the corresponding hydrochloride salts. 1H NMR
(CDCl3): δ 2.63 (3H, s, CH3NH), 2.70 (3H, s, CH 3NH), 3.20
(2H, bs, CH2N), 3.29 (2H, bs, CH2N), 4.16 (2H, bs, CH2O), 4.34
(2H, bs, CH2O), 6.64 (2H, d, J ) 8.4, alkOPhH), 6.85 (2H, d,
J ) 8.4, alkOPhH ), 6.90-7.36 (24H, m, PhH ), 9.73 (4H, bs,
Sch em e 2. Syn th etic P a th w a y to
(E)-r-(Deoxygu a n osin -N2-yl)-N-d esm eth ylta m oxifen
(9)a
2 × CH3NH2+). MS (EI): m/z 409, 407 (M+), 352, 350 (M+
-
CH2CH2NHCH3 + 1), 329 (M+ - Br + 1), 272 (M+ - Br - CH2-
CH2NHCH3 + 2), 252 (M+ - Br - C6H5 + 1), 239 (M+ - Br -
C7H5), 178 {[(C6H5)2C2]+}, 58 [(CH2CH2NHCH3)+]. Anal. Calcd
for C23H22BrNO‚HCl: C, 62.11%; H, 5.21%; N, 3.15%. Found:
C, 62.17%; H, 5.35%; N, 3.04%.
(2) (E)-4-[4-(2-Meth ylam in oeth oxy)ph en yl]-3,4-diph en yl-
bu t-3-en -2-ol (r-Hyd r oxy-N-d esm eth ylta m oxifen , 8). The
synthetic procedure was adapted from the method of Foster
et al. (55). Briefly, (E,Z)-1-bromo-2-[4-(2-methylaminoethoxy)-
phenyl]-1,2-diphenylethene (11, 500 mg, 1.22 mmol) was dis-
solved in 10 mL of freshly distilled dry THF, and the solution
was cooled to -110 °C with liquid nitrogen and a 1/1 mixture
of methanol and ethanol, and kept under argon. An excess of
1.6 M n-butyllithium (4 mL, 5.2 equiv) was added, while the
temperature was kept below -100 °C. Acetaldehyde was then
added dropwise, until the dark brown color of the organolithium
intermediate disappeared and the mixture became pale yellow.
The excess of acetaldehyde was quickly removed by evaporation,
and the mixture was added to water (100 mL). Following
extraction with methylene chloride, the product was separated
from the Z isomer by TLC on silica gel (Merck) by eluting twice
with a methylene chloride/methanol mixture (9/1) and recovered
in 12% yield by precipitation from diethyl ether. Mp: 189-190.5
°C. UV (methanol): λmax 235 (ꢀ ) 18 700 M-1 cm-1), 267 nm (ꢀ
) 10 600 M-1 cm-1). 1H NMR (DMSO-d6): δ 0.98 (3H, d, J )
6.6, CH3CH), 2.24 (3H, s, CH3NH), 2.69 (2H, t, J ) 5.7, CH2N),
3.81 (2H, t, J ) 5.7, CH2O), 4.57 (1H, m, J ) 6.6, J ′ ) 3.0,
CH3CH), 4.70 (1H, d, J ) 3.0, OH), 6.55 (2H, d, J ) 8.4,
alkOPhH), 6.74 (2H, d, J ) 8.4, alkOPhH), 7.10-7.30 (8H, m,
PhH), 7.37 (2H, t, J ) 7.2, PhH). 13C NMR (CDCl3): δ 22.43
(CH3CH), 35.82 (CH3N), 50.40 (CH2N), 66.21 (CH3CH), 68.04
(CH2O), 113.35 (ArCH), 126.54 (ArCH), 126.60 (ArCH), 127.67
(ArCH), 127.91 (ArCH), 129.50 (ArCH), 131.07 (ArCH), 131.41
(ArCH), 134.77 (Cquat), 138.38 (Cquat), 140.50 (Cquat), 141.68
(Cquat), 141.94 (Cquat), 156.86 (Cquat). MS (EI): m/z 373 (M+), 355
(M+ - H2O), 272 (M+ - CH2CH2NHCH3 - CH3CHOH + 2), 240
(M+ - CH2CH2NHCH3 - C6H5 + 2), 183 [(C6H5CHC6H4OH)+],
58 [(CH2CH2NHCH3)+], 44 [(CH2NHCH3)+]. Anal. Calcd for
C25H27NO2‚H2CO3: C, 71.70%; H, 6.71%; N, 3.22%. Found: C,
71.83%; H, 6.55%; N, 3.40%.
(3) (E)-r-(Deoxygu a n osin -N2-yl)-N-d esm eth ylta m oxifen
(9). R-Hydroxy-N-desmethyltamoxifen (8, 21 mg, 56 µmol) and
the SO3‚pyridine complex (41.5 mg, 4.7 equiv) were mixed in
500 µL of dry pyridine. The mixture was stirred for 1 h at 37
°C and then washed twice with 5 mL of diethyl ether. The pellet
was resuspended in 2.5 mL of dry ethanol; the insoluble
materials were removed by centrifugation, and the supernatant
was added in two successive portions to a solution of salmon
sperm DNA (2.24 mg/mL) in 20 mL of 5 mM Bis-Tris and 0.1
mM EDTA (pH 7.1). The mixture was vortexed upon each
addition and then incubated overnight at 37 °C. Nonbonded
materials were removed by extraction with 2 × 1 volume of
n-butanol that had been presaturated with 5 mM Bis-Tris and
0.1 mM EDTA (pH 7.1). The DNA was precipitated by addition
a
(a) (1) ClCO2CH(Cl)CH3, ClCH2CH2Cl, reflux; (2) CH3OH,
reflux. (b) (1) BuLi; (2) CH3CHO. (c) (1) SO3‚pyridine, pyridine,
37 °C; (2) DNA; (3) enzymatic hydrolysis. Both 10 and 11 were
used as mixtures of the E and Z diastereomers.
In str u m en ta tion . The melting temperature of R-hydroxy-
N-desmethyltamoxifen was measured with an Electrothermal
apparatus and is uncorrected.
HPLC analyses were conducted with a µBondapak C18 column
(0.39 cm × 30 cm; Waters Associates, Milford, MA), using either
a Varian system consisting of a Star 9012 ternary gradient
pump and a Polychrom 9065 diode array spectrophotometric
detector (Varian, Inc., Palo Alto, CA), equipped with a Rheodyne
model 7125 injector (Rheodyne, Cotati, CA), or
a Waters
Associates system consisting of two model 510 pumps and a
model 660 automated gradient controller, equipped with a
Rheodyne model 7125 injector and a Hewlett-Packard 1050
diode array spectrophotometric detector (Hewlett-Packard,
Wilmington, DE). Peaks were monitored at 254 or 280 nm.
HPLC analyses of 32P-postlabeled samples were conducted with
a 5 µm Delta-Pak C18-100 column (0.39 cm × 15 cm, Waters
Associates). 32P levels were monitored with
a Radiomatic
Flo-One model A-500 on-line radioactivity detector (Packard
Instruments, Meriden, CT).
UV spectra were recorded with a Beckman DU-40 UV/vis
spectrophotometer.
1H NMR spectra were obtained either on a Varian Unity 300
spectrometer, operating at 300 MHz, or on a Bruker AM500
spectrometer, operating at 500 MHz. 13C NMR spectra were
recorded on the same instruments, operating at 75.4 or 125.7
MHz, respectively. Chemical shifts are reported in parts per
million downfield from tetramethylsilane, and coupling con-
stants are reported in hertz.
Mass spectra were recorded on either a Finnigan TSQ-700
GS/MS system, operated in the electron ionization (EI) mode,
with the sample being introduced via a direct exposure probe
(DEP), or a Finnigan TSQ-7000 LC/MS system, operated in the
electrospray ionization (ESI) mode. For ESI spectral measure-
ments, 50% methanol containing 0.1% ammonium formate (pH
3.5) was used at a flow rate of 0.2 mL/min.
Elemental analyses were performed at the M-H-W Labora-
tories (Phoenix, AZ).
Syn th eses. (1) (E,Z)-1-Br om o-2-[4-(2-m eth ylam in oeth oxy)-
p h en yl]-1,2-d ip h en yleth en e (11; Sch em e 2). (E,Z)-1-Bromo-
2-[4-[2-(dimethylamino)ethoxy]phenyl]-1,2-diphenylethene (55)
(10, 3 g, 7.1 mmol, 1/1 E/Z) was dissolved in 1,2-dichloroethane
(40 mL), and the solution was cooled in an ice bath. A solution
of 1-chloroethyl chloroformate (2 mL, 18.5 mmol) in 1,2-di-
chloroethane (10 mL) was added dropwise over a period of 5

Major Adduct from R-Hydroxy-N-desmethyltamoxifen
Chem. Res. Toxicol., Vol. 13, No. 3, 2000 203
Ta ble 1. 1H NMR Da ta for (E)-r-(Deoxygu a n osin -N2-yl)-N-d esm eth ylta m oxifen
δ (ppm)a
N-desmethyltamoxifen fragment
deoxyribose fragment
guanine fragment
1.26b (3H, d, J ) 7.0, CH3CH)
2.31 (1H, m, H2′′)
2.52 (1H, m, H2′)
3.50 (1H, m, H5′/H5′′)
3.54 (1H, m, H5′/H5′′)
3.86 (1H, m, H4′)
5.99c (1H, d, J ) 7.4, N2H)
7.97 (1H, s, H8)
1.85c (1H, bs, CH3NH)
2.24 (3H, s, CH3NH)
10.08c (1H, bs, N1H)
2.68 (2H, t, J ) 5.6, CH2N)
3.80 (2H, t, J ) 5.6, CH2O)
4.89 (1H, m, CH3CH)
4.37 (1H, m, H3′)
6.53d (2H, d, J ) 8.8, alkOPhH)
6.77d (2H, d, J ) 8.8, alkOPhH)
7.03 (2H, d, J ) 6.9, PhH)
7.16 (1H, t, J ) 7.3, PhH)
7.22e (2H, t, J ) 7.3, PhH)
7.32 (1H, t, J ) 7.5, PhH)
7.41 (2H, d, J ) 6.9, PhH)
7.49f (2H, t, J ) 7.6, PhH)
4.92c (1H, t, J ) 5.2, 5′-OH)
5.34c (1H, d, J ) 3.3, 3′-OH)
6.18 (1H, t, J ) 6.2, H1′)
a
b
d
Recorded in DMSO-d6. Abbreviations: b, broad; d, doublet; m, multiplet; s, singlet; t, triplet. c Exchangeable with D2O. Protons
from the alkoxyphenyl ring. e Coupled to the signals at 7.03 and 7.16 ppm. f Coupled to the signals at 7.32 and 7.41 ppm.
of 5 M NaCl (0.1 volume) and ethanol (3 volumes), washed with
cold 70% ethanol (2 × 20 mL), and redissolved in 20 mL of 5
mM Bis-Tris and 0.1 mM EDTA (pH 7.1). The modified DNA
was hydrolyzed to nucleosides by treatment with DNAse I,
followed by alkaline phosphatase and phosphodiesterase I (56).
The adducts were then partitioned into n-butanol (6 × 10 mL),
which had been presaturated with 5 mM Bis-Tris and 0.1 mM
EDTA (pH 7.1), and the butanol extracts were combined and
back-extracted with 5 mM Bis-Tris and 0.1 mM EDTA (pH 7.1),
presaturated with n-butanol. After the butanol was evaporated,
the residue was redissolved in methanol (2.5 mL) and purified
by HPLC, at a flow rate of 2 mL/min, using a 17 min linear
gradient of 0 to 60% acetonitrile in 100 mM ammonium acetate
(pH 5.7), followed by a 3 min linear gradient to 100% acetonitrile
and a 5 min isocratic elution with acetonitrile. The major adduct,
(E)-R-(deoxyguanosin-N2-yl)-N-desmethyltamoxifen (9), eluted
100% solvent B. The flow rate was 1 mL/min. To avoid
interference from the high amounts of radioactivity associated
with free 32P, unreacted [γ-32P]ATP, and 32P-postlabeled normal
nucleotides, the eluent from the first 15 min was diverted from
the detector.
Resu lts a n d Discu ssion
Syn t h esis of r-H yd r oxy-N-d esm et h ylt a m oxifen
(8). The bromoolefin (10), which was obtained as a
mixture of the E and Z diastereomers (55), was the
starting material for the synthesis of R-hydroxy-N-
desmethyltamoxifen. Quantitative N-demethylation of
the tertiary amino group to 11 (Scheme 2) was easily
achieved by reaction with 1-chloroethyl chloroformate,
followed by methanolysis of the intermediate carbamate
using the methodology of Olofson et al. (60). Although
no attempt was made to separate the E and Z isomers
of 11, which were obtained as the corresponding hydro-
chloride salts, the 1H NMR and mass spectral data
(cf. Materials and Methods) confirmed the assigned
structure.
The synthesis of R-hydroxy-N-desmethyltamoxifen (8)
was adapted from the procedure described by Foster
et al. for R-hydroxytamoxifen (55). Specifically, the
(E,Z)-N-demethylated bromoolefin (11) was treated with
approximately 5 equiv of n-butyllithium at -110 °C,
followed by reaction of the alkenyllithium intermediate
with acetaldehyde (Scheme 2). This methodology had
potential difficulties in this particular case, due to the
presence of a labile proton in the secondary amino group
of 11. We reasoned that using an excess of n-butyllithium
and a temperature sufficiently low to minimize secondary
reactions, it would be possible to generate 8. In fact,
despite competitive deprotonation of the amino group,
which was accompanied by loss of N-methylaziridine,
presumably through an internal SN2 reaction, R-hydroxy-
N-desmethyltamoxifen was obtained in 12% yield after
separation from the dealkylated products and Z isomer
by TLC. Spectroscopic data (1H and 13C NMR and MS;
cf. Materials and Methods) of the isolated product were
fully consistent with addition of an R-hydroxyethyl frag-
ment to the triarylethylene structure and with the
presence of an intact N-methylaminoethoxy substituent.
The assignment of the E configuration was based on the
1H NMR spectrum, where an upfield shift of the AB
quartet corresponding to the protons of the alkoxyphenyl
ring (δ 6.55 and 6.74), reflected the combined shielding
effect of the two phenyl rings (61, 62).
1
at 14.3 min. For the H NMR data, see Table 1. MS (ESI): m/z
+
623 (MH+). MS/MS (m/z 623): m/z 507 (MH2 - dR), 356
(M+ - dG), 330 (MH2+ - dG - CH3CH), 178 [(Gua + CHCH3)+],
58 [(CH2CH2NHCH3)+].
Tr ea tm en t of An im a ls. Rats were treated with tamoxifen
and R-hydroxy-N-desmethyltamoxifen according to the protocol
of White et al. (13). Specifically, female Sprague-Dawley rats
(four per group, 8 weeks old, obtained from the breeding colony
at the National Center for Toxicological Research) were treated
by gavage with seven daily doses of tamoxifen (20 mg/kg, 54
µmol/kg, dissolved in 200 µL of trioctanoin). Four additional rats
were treated in the same manner with equimolar doses of
R-hydroxy-N-desmethyltamoxifen (20.1 mg/kg, dissolved in 200
µL of trioctanoin), and four control animals were treated with
200 µL of trioctanoin alone. Twenty-four hours after the last
treatment, the animals were killed by exposure to carbon
dioxide. Hepatic nuclei were isolated by the method of Basler
et al. (57), and DNA was prepared from the nuclei as described
by Beland et al. (58).
32P -P ostla belin g An a lyses. 32P-Postlabeling analyses were
conducted by the nuclease P1 enrichment procedure of Reddy
and Randerath (59), essentially as described by Beland et al.
(36). Briefly, 10 µg of DNA was hydrolyzed with micrococcal
endonuclease and spleen phosphodiesterase for 3 h at 37 °C and
then treated for 1 h with nuclease P1. After evaporation in a
Speed-Vac concentrator, each sample was resuspended in water
and labeled with 20 µCi of carrier-free [γ-32P]ATP in the
presence of PNK in a total volume of 20 µL.
Aliquots of each labeling mixture were analyzed by HPLC.
The adducts were separated as follows: from 0 to 10 min,
isocratic elution with 58% solvent A [1.2 M ammonium formate
and 10 mM ammonium phosphate (pH 4.5)] and 42% solvent B
[24% acetonitrile in 1.2 M ammonium formate and 10 mM
ammonium phosphate (pH 4.5)]; from 10 to 20 min, a linear
gradient to 83% solvent B; from 20 to 40 min, isocratic elution
with 83% solvent B; and from 40 to 60 min, a linear gradient to

204 Chem. Res. Toxicol., Vol. 13, No. 3, 2000
Gamboa da Costa et al.
F igu r e 2. ESI MS/MS fragmentation pattern of the MH+ ion
from (E)-R-(deoxyguanosin-N2-yl)-N-desmethyltamoxifen (9).
The assignments are discussed in the text.
F igu r e 1. HPLC of the enzymatic hydrolysate obtained from
reacting R-sulfoxy-N-desmethyltamoxifen with DNA. The elu-
tion conditions are outlined in Materials and Methods. The
absorbance was monitored at 254 nm. The inset shows the UV
absorption spectrum of the major peak (1), as detected by HPLC
in 100 mM ammonium acetate (pH 5.7)/acetonitrile.
Syn th esis a n d Isola tion of th e Ma jor DNA Ad d u ct
(9). R-Hydroxy-N-desmethyltamoxifen (8) was further
activated by reaction with the SO3‚pyridine complex
using the methodology described by Dasaradhi and
Shibutani for R-sulfoxytamoxifen (34). Since R-sulfoxy-
N-desmethyltamoxifen was anticipated to be unstable
upon exposure to the atmosphere, it was reacted with
DNA immediately after being produced and spectroscopic
characterization of the intermediate was not attempted.
After enzymatic hydrolysis of the modified DNA to
nucleosides, the HPLC profile of the n-butanol extract
(Figure 1) indicated the presence of one major (1) and at
least two minor (2 and 3) peaks eluting at 12-17 min.
These were presumably DNA adducts since they were
not detected in control incubations. The major peak (1),
with a retention time of 14.3 min, was isolated and
F igu r e 3. Aromatic region of the 1H NMR spectrum of (E)-R-
(deoxyguanosin-N2-yl)-N-desmethyltamoxifen (9), recorded in
DMSO-d6. Full chemical shift assignments are presented in
Table 1.
1
characterized by MS and H NMR (vide infra). The UV
spectrum of the adduct exhibited a maximum at ca. 250
nm and a shoulder at ca. 280 nm (Figure 1, inset), closely
resembling those reported for N2-deoxyguanosyl adducts
from R-acetoxy-, R-sulfoxy-, and 4-hydroxytamoxifen (33-
35, 42), which suggested the presence of essentially the
same chromophore. The minor peaks (2 and 3) were not
isolated, but their UV spectra (not shown) were almost
identical to that of peak 1, which suggests that peaks 2
and 3 were diastereomers of the major product.
Ch a r a cter iza tion of th e Isola ted Ad d u ct (9). (1)
Ma ss Sp ectr u m . ESI mass spectrometry of the isolated
product in the positive ion mode yielded an ion at m/z
623 (C35H39N6O5), which corresponded to a protonated
adduct of N-desmethyltamoxifen and deoxyguanosine. A
product ion analysis of the protonated molecular ion was
performed in the ESI MS/MS mode (Figure 2). Prominent
fragments were found at m/z 507 (52%), 356 (100%), and
330 (74%), and additional minor ions at m/z 178 (14%)
and 58 (3%). The detection of strong ions at m/z 507
(MH+ - 116) and 356 (MH+ - 267) indicated that loss
of deoxyribose and deoxyguanosine, respectively, were
major fragmentation pathways for the adduct. The pres-
ence of the minor ion at m/z 58 [N-methylaziridinium,
(CH2CH2NHCH3)+] showed that the N-methylamino-
ethoxy substituent was intact in the parent molecule. The
most informative fragments for structural elucidation
were the ions at m/z 330 [(N-desmethyltamoxifen - CH3-
CH + 1)+] and 178 [(guanine + CH3CH)+], which were
fully consistent with attachment of deoxyguanosine to the
allylic carbon of the N-desmethyltamoxifen segment. This
fragmentation pattern is virtually identical to that of the
N-demethylated adduct detected in rat liver DNA by
Rajaniemi et al. (39), which gives further support to the
significance of R-hydroxy-N-desmethyltamoxifen as a
DNA adduct precursor in vivo.
1
(2) H NMR Sp ectr u m . Conclusive evidence for the
structure of the isolated adduct was obtained by 1H NMR.
The proton chemical shifts for the adduct in DMSO-d6
are summarized in Table 1. Assignments were based on
comparison to the spectra of dG and R-hydroxy-N-
desmethyltamoxifen, combined with homonuclear decou-
pling experiments and chemical exchange of the labile
protons with D2O. All the protons of the N-desmethyl-
tamoxifen and dG moieties were detected. Elucidation
of the site of attachment of dG to the N-desmethyl-
tamoxifen segment stemmed from the presence of the
imino and H8 protons of dG at δ 10.08 and 7.97, re-
spectively, while the exocyclic amino group accounted for
only one proton at δ 5.99 (Table 1). This proton was split
into a doublet (Figure 3) and coupled to the methine
multiplet at δ 4.89, which indicated that substitution
occurred at the exocyclic nitrogen of dG through the
allylic carbon of N-desmethyltamoxifen. Furthermore,
two upfield, mutually coupled, aromatic doublets were
present at δ 6.53 and 6.77 and assigned to the alkoxy-
phenyl ring protons. The position of these resonances

Major Adduct from R-Hydroxy-N-desmethyltamoxifen
Chem. Res. Toxicol., Vol. 13, No. 3, 2000 205
incorporated into peaks 1-3, tamoxifen and R-hydroxy-
N-desmethyltamoxifen gave similar levels of hepatic
DNA adducts.
When assessed by 32P-postlabeling, DNA modified
in vitro with R-sulfoxy-N-desmethyltamoxifen had one
major (2) and three minor adducts (1, 3, and 4, Figure
4c). The major adduct present in the in vitro modified
DNA standard, which was identified as the 3′,5′-bis-
phosphate of (E)-R-(deoxyguanosin-N2-yl)-N-desmethyl-
tamoxifen (vide supra), had the same retention time as
peak 2 detected in liver DNA from tamoxifen- and
R-hydroxy-N-desmethyltamoxifen-treated rats. Upon
mixed injections (not shown), peak 2 from the in vivo
samples co-eluted with the major adduct (2) in the
standard. In addition to the co-identity of peak 2, one of
the minor adducts detected in the DNA standard had a
retention time (27.8 min, Figure 4c) virtually identical
to that of peak 1 (panels a and b of Figure 4), while
another minor peak (retention time of 29.9 min, Figure
4c) co-eluted with peak 3. These results suggest that
peaks 1 and 3 from liver DNA of rats treated with
R-hydroxy-N-desmethyltamoxifen might be diastereo-
mers of (E)-R-(deoxyguanosin-N2-yl)-N-desmethyltamox-
ifen. It should be noted, however, that previous studies
(34, 35) have shown the reaction of R-acetoxytamoxifen
with DNA in vitro yields diastereomers of (E)-R-(deoxy-
guanosin-N2-yl)tamoxifen as minor adducts. Since these
adducts have retention times similar to those of peaks 1
and 2, it is also possible that a portion of peaks 1 and 2
in the livers of rats treated with tamoxifen may arise
from R-hydroxytamoxifen.
F igu r e 4. Representative HPLC profiles of liver DNA from
female Sprague-Dawley rats treated with (a) tamoxifen or (b)
R-hydroxy-N-desmethyltamoxifen and (c) DNA modified in vitro
with R-sulfoxy-N-desmethyltamoxifen. The 32P-postlabeling and
elution conditions are outlined in Materials and Methods;
approximately 10 µg of DNA was postlabeled in each instance.
Con clu sion s
Recent data from rats treated with N-desmethyl-
tamoxifen have indicated that this metabolite gives rise
to one of the major adducts detected in the livers of rats
treated with tamoxifen (37, 38). Our results confirm that
this major N-demethylated adduct (peak 2, Figure 4)
results from hydroxylation of N-desmethyltamoxifen at
the allylic carbon to yield R-hydroxy-N-desmethyltamox-
ifen. The work by the Phillips (37) and Martin (38) groups
has also suggested that peak 1 (Figure 4a) originated
from N,N-didesmethyltamoxifen. As shown in Figure 4b,
the liver DNA of rats treated with R-hydroxy-N-des-
methyltamoxifen had a minor adduct peak with the same
retention time as peak 1. In addition, one of the minor
adducts present in the synthetic standard (Figure 4c) had
the same retention time as peak 1. This suggests that
the adduct corresponding to peak 1 in vivo may not arise
from N,N-didesmethyltamoxifen but rather is a minor
adduct of N-desmethyltamoxifen. Additional work is
needed to determine the precise metabolic pathway
leading to this minor adduct. Our data also indicate
that a portion of the adducts previously attributed to
R-hydroxytamoxifen (peak 3) may actually be due to
R-hydroxy-N-desmethyltamoxifen.
indicated that the protons were shielded by a combined
effect of the two phenyl substituents (61, 62), which
confirmed that the N-desmethyltamoxifen segment was
in a trans configuration. Therefore, the major adduct
from reaction of R-sulfoxy-N-desmethyltamoxifen with
DNA was characterized as (E)-R-(deoxyguanosin-N2-yl)-
N-desmethyltamoxifen (Scheme 2). We could not estab-
lish if this adduct was obtained as a single product or a
mixture of epimers at the allylic carbon.
DNA Ad d u ct An a lyses in R a t s Tr ea t ed w it h
Ta m oxifen or r-Hyd r oxy-N-d esm eth ylta m oxifen .
With the aim of establishing the significance of R-
hydroxy-N-desmethyltamoxifen as a DNA adduct pre-
cursor in vivo, female Sprague-Dawley rats were treated
by gavage daily for 7 days with tamoxifen, R-hydroxy-
N-desmethyltamoxifen, or the solvent alone. The hepatic
DNA adducts formed in vivo were analyzed by 32P-
postlabeling in combination with HPLC.
Only background levels of adducts were detected in the
liver DNA of control rats (not shown). Fully consistent
with previous reports (33, 36-38), one minor and two
major DNA adducts, with retention times of 27.7, 28.8,
and 29.9 min (peaks 1-3, respectively, Figure 4a), were
found in liver DNA from rats treated with tamoxifen.
Peak 3 has been identified as R-(deoxyguanosin-N2-yl)-
tamoxifen on the basis of coelution with the synthetic
standard (33, 36-38). Liver DNA from rats treated with
R-hydroxy-N-desmethyltamoxifen had one major and two
minor adducts which coeluted, respectively, with peaks
2, 1, and 3 present in liver DNA from tamoxifen-treated
rats (Figure 4b). On the basis of the amount of 32P
Ack n ow led gm en t. We thank J . Pat Freeman and
J ackson O. Lay, J r., for the mass spectra, Richard Beger
for some of the NMR spectra, and Cindy Hartwick
for helping to prepare the manuscript. This work was
supported in part by a Postgraduate Research Program
administered by the Oak Ridge Institute for Science and
Education, by a research grant from Program PRAXIS
XXI, Fundac¸a˜o para a Cieˆncia e Tecnologia (FCT),
Portugal, and by a fellowship to G.G.C. from Sub-
programa Cieˆncia e Tecnologia, FCT, Portugal.

206 Chem. Res. Toxicol., Vol. 13, No. 3, 2000
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