Quantification of tamoxifen DNA adducts using on-line sample preparation and HPLC-electrospray ionization tandem mass spectrometry

Article abstract of DOI:10.1021/tx020090g

The nonsteroidal antiestrogen tamoxifen is used as an adjuvant chemotherapeutic agent for the treatment of all stages of hormone-dependent breast cancer and more recently as a chemopreventive agent in women with elevated risk of developing the disease. While clearly beneficial for the treatment of breast cancer, tamoxifen has been reported to increase the risk of endometrial cancer in women. Furthermore, it has been shown to be hepatocarcinogenic in rats. Tamoxifen is clearly genotoxic in rat liver, as indicated by the formation of DNA adducts; the occurrence of tamoxifen DNA adducts in human endometrial tissue is more controversial. The detection and quantitation of tamoxifen DNA adducts have relied primarily upon ³²P-postlabeling, with other techniques, such as immunoassays and accelerator mass spectrometry, being used to a much lesser extent. To expand the range of available analytical methodologies for quantifying tamoxifen DNA adducts, we have developed an assay using on-line sample preparation, coupled with HPLC and electrospray ionization tandem mass spectrometry (ES-MS/MS). α-Acetoxytamoxifen was reacted with salmon testis DNA at ratios between 0.1 ng and 1 mg α-acetoxytamoxifen per mg DNA. After enzymatic hydrolysis to nucleosides, the most highly modified DNA samples were analyzed by HPLC-UV, which indicated the presence of two adduct peaks in approximately a 1:4 ratio. The major adduct was isolated, rigorously characterized as (E)-α-(deoxyguanosin-N²-yl)tamoxifen, and quantified on the basis of its molar extinction coefficient. A similar reaction was conducted with [N(CD₃)₂]-α-acetoxytamoxifen to prepare a deuterated adduct that could serve as an internal standard for ES-MS/MS. The limit of detection for the HPLC-ES-MS/MS method was approximately 5 adducts/10⁹ nucleotides, with an intra- and interassay precision of 3% relative standard deviation. The method was validated over the range of 8-1 520 000 adducts/10⁸ nucleotides using 100 μg samples of DNA modified in vitro. Analysis of liver DNA from female Sprague - Dawley rats treated by gavage with seven daily doses of 20 mg tamoxifen/kg body weight gave a value of 496 ± 16 adducts/10⁸ nucleotides for (E)-α-(deoxyguanosin-N²-yl)tamoxifen and 626 ± 18 adducts/10⁸ nucleotides for (E)-α-(deoxyguanosin-N²-yl)-N-desmethyltamoxifen. These data indicate that the HPLC-ES-MS/MS methodology has sufficient sensitivity and precision to be useful in the analysis of tamoxifen DNA adducts formed in vivo in experimental models and may be able to detect tamoxifen DNA adduct formation in human tissue samples.

Full text of DOI:10.1021/tx020090g

Chem. Res. Toxicol. 2003, 16, 357-366
357
Qu a n tifica tion of Ta m oxifen DNA Ad d u cts Usin g
On -Lin e Sa m p le P r ep a r a tion a n d HP LC-Electr osp r a y
Ion iza tion Ta n d em Ma ss Sp ectr om etr y
Gonc¸alo Gamboa da Costa,† M. Matilde Marques,*,† Frederick A. Beland,‡
J ames P. Freeman,§ Mona I. Churchwell,‡ and Daniel R. Doerge*,‡
Centro de Qu´ımica Estrutural, Complexo I, Instituto Superior Te´cnico, Av. Rovisco Pais,
1049-001 Lisboa, Portugal, and Divisions of Biochemical Toxicology and Chemistry,
National Center for Toxicological Research, J efferson, Arkansas 72079
Received October 7, 2002
The nonsteroidal antiestrogen tamoxifen is used as an adjuvant chemotherapeutic agent
for the treatment of all stages of hormone-dependent breast cancer and more recently as a
chemopreventive agent in women with elevated risk of developing the disease. While clearly
beneficial for the treatment of breast cancer, tamoxifen has been reported to increase the risk
of endometrial cancer in women. Furthermore, it has been shown to be hepatocarcinogenic in
rats. Tamoxifen is clearly genotoxic in rat liver, as indicated by the formation of DNA adducts;
the occurrence of tamoxifen DNA adducts in human endometrial tissue is more controversial.
The detection and quantitation of tamoxifen DNA adducts have relied primarily upon 32P-
postlabeling, with other techniques, such as immunoassays and accelerator mass spectrometry,
being used to a much lesser extent. To expand the range of available analytical methodologies
for quantifying tamoxifen DNA adducts, we have developed an assay using on-line sample
preparation, coupled with HPLC and electrospray ionization tandem mass spectrometry (ES-
MS/MS). R-Acetoxytamoxifen was reacted with salmon testis DNA at ratios between 0.1 ng
and 1 mg R-acetoxytamoxifen per mg DNA. After enzymatic hydrolysis to nucleosides, the
most highly modified DNA samples were analyzed by HPLC-UV, which indicated the presence
of two adduct peaks in approximately a 1:4 ratio. The major adduct was isolated, rigorously
characterized as (E)-R-(deoxyguanosin-N2-yl)tamoxifen, and quantified on the basis of its molar
extinction coefficient. A similar reaction was conducted with [N(CD3)2]-R-acetoxytamoxifen to
prepare a deuterated adduct that could serve as an internal standard for ES-MS/MS. The
limit of detection for the HPLC-ES-MS/MS method was approximately 5 adducts/109 nucleo-
tides, with an intra- and interassay precision of 3% relative standard deviation. The method
was validated over the range of 8-1 520 000 adducts/108 nucleotides using 100 µg samples of
DNA modified in vitro. Analysis of liver DNA from female Sprague-Dawley rats treated by
gavage with seven daily doses of 20 mg tamoxifen/kg body weight gave a value of 496 ( 16
adducts/108 nucleotides for (E)-R-(deoxyguanosin-N2-yl)tamoxifen and 626 ( 18 adducts/108
nucleotides for (E)-R-(deoxyguanosin-N2-yl)-N-desmethyltamoxifen. These data indicate that
the HPLC-ES-MS/MS methodology has sufficient sensitivity and precision to be useful in the
analysis of tamoxifen DNA adducts formed in vivo in experimental models and may be able to
detect tamoxifen DNA adduct formation in human tissue samples.
and Drug Administration to approve the use of tamoxifen
as a prophylactic agent against breast cancer in high-
In tr od u ction
The nonsteroidal antiestrogen tamoxifen1 (1, Figure 1)
has been used for nearly three decades as an adjuvant
chemotherapeutic agent for the treatment of all stages
of hormone-dependent breast cancer (1). More recently,
data from a large-scale clinical trial in the United States
have shown that the administration of tamoxifen to
women at high risk of developing the disease resulted in
a 50% reduction in the occurrence of invasive breast
cancer (2). This observation has prompted the U.S. Food
1
Abbreviations: AlkOPh, alkoxyphenyl; AMS, accelerator mass
spectrometry; Bis-Tris, bis(2-hydroxyethyl)iminotris(hydroxymethyl)-
methane; Cquat, quaternary carbon; CIA, chemiluminescence immu-
noassay; DELFIA, dissociation-enhanced lanthanide fluoroimmunoas-
say; N-desmethyltamoxifen, (Z)-1-[4-(2-methylamino)ethoxy]phenyl-
1,2-diphenyl-but-1-ene; dG, 2′-deoxyguanosine; dG-desMeTam, (E)-R-
(deoxyguanosin-N2-yl)-N-desmethyltamoxifen; dG-Tam, (E)-R-(deoxy-
guanosin-N2-yl)tamoxifen; dG-Tam-d6, (E)-R-(deoxyguanosin-N2-yl)-
N,N-bis(trideuteriomethyl)tamoxifen; dR, 2′-deoxyribosyl; EI, electron
ionization; ES, electrospray ionization; FAB, fast atom bombardment;
HRMS, high-resolution mass spectrometry; R-hydroxy-N-desmethyl-
tamoxifen, (E)-4-[4-[2-(methylamino)ethoxy]phenyl]-3,4-diphenylbut-
3-en-2-ol; R-hydroxytamoxifen, (E)-4-[4-[2-(dimethylamino)ethoxy]phen-
yl]-3,4-diphenylbut-3-en-2-ol; R-hydroxytamoxifen-d6, (E)-4-[4-[2-(bis(tri-
deuteriomethyl)amino)ethoxy]phenyl]-3,4-diphenyl-but-3-en-2-ol; LOD,
limit of detection (S/N ) 3); LOQ, limit of quantitation (S/N ) 10);
MRM, multiple reaction monitoring; MS/MS, tandem mass spectrom-
etry; RSD, relative standard deviation (SD/mean × 100); S/N, signal-
to-noise ratio; tamoxifen, (Z)-1-[4-(2-dimethylamino)ethoxy]phenyl-1,2-
diphenylbut-1-ene.
* To whom correspondence should be addressed. M.M.M.: Tel: 351-
21-841-9200. Fax: 351-21-846-4455. E-mail: matilde.marques@ist.utl.pt.
D.R.D.: Tel: (870)543-7943. Fax: (870)543-7720. E-mail: ddoerge@
nctr.fda.gov.
† Instituto Superior Te´cnico.
‡ Division of Biochemical Toxicology, National Center for Toxicologi-
cal Research.
§ Division of Chemistry, National Center for Toxicological Research.
10.1021/tx020090g CCC: $25.00 © 2003 American Chemical Society
Published on Web 02/11/2003

358 Chem. Res. Toxicol., Vol. 16, No. 3, 2003
Gamboa da Costa et al.
43). Some additional observations consistent with tamox-
ifen genotoxicity include the induction of aneuploidy and
chromosomal aberrations in rat liver and of micronuclei
in metabolically proficient human cells (reviewed in 5).
Furthermore, tamoxifen has been associated with muta-
tions in the p53 tumor supressor gene of rat hepatocar-
cinomas (44), and both tamoxifen and R-hydroxytamox-
ifen have been shown to cause mutations in the liver lacI
and cII genes of Big Blue transgenic rats (45-50).
Similarly to what has been shown in vitro for all of the
dG-Tam diastereomers (51), these mutations were pre-
dominantly G f T transversions.
With few exceptions (52, 53), most studies have failed
to detect DNA adducts in the uterus and other extrahe-
patic tissues from rats administered tamoxifen or tamox-
ifen derivatives (18, 23, 24, 29, 54). These results,
combined with the lack of detectable mutations in ex-
trahepatic tissues (29, 46, 47, 49), have suggested that
tamoxifen-induced endometrial tumors in the rat do not
arise from a genotoxic pathway. Although very few
studies have been conducted in other animal models,
recent preliminary reports of the detection of tamoxifen
DNA adducts in various tissues of female cynomologous
monkeys, including the uterus (55, 56), support the
existence of interspecies variations in tamoxifen activa-
tion.
F igu r e 1. Structures of tamoxifen (1) and its metabolites
N-desmethyltamoxifen (2), R-hydroxytamoxifen (3), and R-hy-
droxy-N-desmethyltamoxifen (4).
The evidence for uterine DNA damage in women on
tamoxifen therapy has been the focus of intense debate.
Using the 32P-postlabeling methodology, Hemminki et al.
(57) were the first to report the detection of DNA adducts
in endometrial samples from women administered tamox-
ifen, at levels of 2.7 adducts/109 nucleotides, but their
study has been criticized, due to high background levels
and the lack of adduct standards (58). Using an improved
32P-postlabeling procedure, coupled with HPLC detection,
Shibutani and colleagues corroborated the detection of
tamoxifen DNA adducts in the endometrial tissue of
patients administered tamoxifen (59, 60). The adducts
were identified on the basis of co-chromatography with
synthetic standards and were estimated to be in the
range of 0.2-18 adducts/108 nucleotides. However, using
a similar approach, Carmichael, Phillips, and co-workers
found no evidence for tamoxifen binding to human
endometrial DNA (61, 62). In a preliminary study, Martin
et al. used the extremely sensitive technique of AMS to
assay uterine DNA following a single dose of [14C]-
tamoxifen to women prior to surgery (63). Radiolabeled
carbon was detected in the uterus of the treated patients,
but the evidence for DNA damage was not conclusive.
As indicated above, the 32P-postlabeling technique has
been, in most instances, the method of choice for the
detection and quantitation of tamoxifen DNA adducts
formed in vitro and in vivo, with a few studies reporting
the use of AMS (53, 63) or immunoassays (55, 64).
Despite their sensitivity, each of these techniques has a
range of different limitations, particularly concerning the
unequivocal characterization of the adducts (reviewed in
65). In view of the controversy surrounding the detection
of tamoxifen DNA adducts in human endometrial samples,
the availability of analytical methodologies capable of
measuring, with high chemical specificity, small amounts
of tamoxifen-derived adducts in target tissues is clearly
required. In the present study, we describe the develop-
ment and validation of an on-line method that involves
HPLC separation and ES-MS/MS detection, using a
deuterated adduct as internal standard and MRM. The
F igu r e 2. Structures of (E)-R-(deoxyguanosin-N2-yl)tamoxifen
(dG-Tam; 5) and (E)-R-(deoxyguanosin-N2-yl)-N-desmethylta-
moxifen (dG-desMeTam; 6), the major adducts formed in the
livers of tamoxifen-treated rats.
risk women. Despite this beneficial effect regarding
breast cancer, a major cause of concern is the increased
incidence of endometrial cancer among women adminis-
tered tamoxifen (1, 2). In particular, suggestions that
long-term tamoxifen users develop endometrial cancers
with a worse prognosis than that of sporadic endometrial
cancers (3, 4) raise questions about the net benefit from
the prophylactic use of the drug by healthy women. The
mechanisms underlying the induction of endometrial
tumors by tamoxifen have yet to be established, although
both the genotoxic (reviewed in 5) and the hormonal (6-
10) properties of the drug have been implicated.
Tamoxifen is a strong hepatocarcinogen in rats (11-
13) and has been shown to cause endometrial tumors in
rats and mice after transplacental, neonatal, or chronic
exposure (14-18). Substantial evidence for a genotoxic
mechanism in rat and mouse liver stems from the
detection of hepatic DNA adducts following dosing regi-
mens with tamoxifen (12, 19-23) and some of its me-
tabolites, such as N-desmethyltamoxifen, R-hydroxyta-
moxifen, and R-hydroxy-N-desmethyltamoxifen (2-4,
Figure 1) (24-30). A considerable body of evidence has
indicated that metabolic activation to DNA binding
electrophiles in rat liver occurs primarily through cyto-
chrome P450-catalyzed hydroxylation at the allylic (R)
carbon of tamoxifen (and presumably 2) (31), followed by
O-esterification, mainly through sulfotransferase-cata-
lyzed sulfation (32-35). The major DNA adducts arising
from these metabolic pathways have been identified as
dG-Tam (5, Figure 2) and dG-desMeTam (6, Figure 2)
by using spectroscopic techniques and through compari-
son with fully characterized synthetic standards (28, 36-

HPLC-ES-MS/ MS Quantification of Tamoxifen DNA Adducts
Chem. Res. Toxicol., Vol. 16, No. 3, 2003 359
F igu r e 3. Synthesis of deuterium-labeled R-hydroxytamoxifen (12). Compounds 8-11 were obtained as mixtures of the E and Z
isomers; compound 12 was separated from the Z isomer by TLC.
Winsford, U.K.), operated in the FAB mode, with the sample
dispersed in a matrix of 3-nitrobenzyl alcohol, a Finnigan TSQ-
method is applied to DNA modified in vitro with R-ace-
toxytamoxifen and from tissues of experimental animals
700 GS/MS system (Thermo Finnigan, San J ose, CA), operated
administered tamoxifen.
in the EI mode, with the sample being introduced via a direct
exposure probe, or a Finnigan TSQ-7000 LC/MS system, oper-
Ma ter ia ls a n d Meth od s
ated in the ES mode. For ES spectral measurements, 50%
methanol containing 0.1% ammonium formate (pH 3.5) was
used at a flow rate of 0.2 mL/min. HRMS was performed at the
Instituto Tecnolo´gico e Nuclear, Sacave´m, Portugal. Elemental
analyses were performed at the M-H-W Laboratories (Phoenix,
Ca u tion : Tamoxifen and its derivatives are potentially geno-
toxic and should be handled with proper care.
Ch em ica ls. Tamoxifen, salmon testis DNA, Bis-Tris, trioc-
tanoin, and the enzymes used in DNA hydrolysis were pur-
AZ) or at the Analytical Laboratory, Instituto Superior Te´cnico,
chased from Sigma Chemical Co. (St. Louis, MO). All other
Lisboa, Portugal.
commercially available reagents were obtained from Aldrich
Syn th eses. R-Hydroxytamoxifen (3) was synthesized by the
method of Foster et al. (67) and converted to R-acetoxytamoxifen
as described by Osborne et al. (36). R-Hydroxy-N-desmethylta-
moxifen (4) was synthesized and converted into its sulfate ester
derivative as described in Gamboa da Costa et al. (28). R-Hy-
droxytamoxifen-d6 (12, Figure 3) was synthesized by the series
of reactions outlined below.
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 (66).
Gen er a l In str u m en ta tion . Melting temperatures were
measured with a Leica Galen III hot-stage apparatus and are
uncorrected. HPLC analyses of tamoxifen derivatives and their
synthetic precursors 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 spectropho-
tometric 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 Co.,
Palo Alto, CA). The peaks were monitored at 254 or 280 nm.
UV spectra were recorded with either a Beckman DU-40 UV/
vis (Beckman Coulter, Fullerton, CA) or a Shimadzu 1202 UV/
vis (Shimadzu Europe, Duisburg, Germany) spectrophotometer.
1H NMR spectra were obtained either on a Varian Unity 300
spectrometer (Varian Deutschland Gmbh, Darmstadt, Ger-
many), operating at 300 MHz, or a Bruker AM500 spectrometer
(Bruker Instruments, Inc., Billerica, MA), operating at 500 MHz.
13C NMR spectra were recorded on the Varian Unity 300
instrument, operating at 75.4 MHz. Chemical shifts are reported
in ppm downfield from tetramethylsilane, and coupling con-
stants are reported in Hz.
(()-[1-(4-Hyd r oxyp h en yl)]-1,2-d ip h en ylet h a n ol (7). A
solution of benzyl bromide (26 mL, 214 mmol) in dry THF (50
mL) was added dropwise, over a period of 1 h, to a refluxing
suspension of magnesium powder (11 g, 452 mmol) in dry THF
(100 mL). The mixture was further refluxed for 20 min and then
allowed to reach room temperature. 4-Hydroxybenzophenone (10
g, 49.4 mmol) in THF (20 mL) was subsequently added over 5
min, and the mixture was stirred for 1 h. The dark green
solution was then quickly filtered to remove excess magnesium,
the magnesium was washed with THF (20 mL), and the
combined THF solutions were poured into a saturated am-
monium chloride solution (800 mL). The product was extracted
with methylene chloride (3 × 1 vol), and the organic extracts
were combined and dried with anhydrous sodium sulfate. The
solvent was evaporated under vacuum, and the crude residue
was washed repeatedly with n-hexane to remove 1,2-diphenyl-
ethane. The carbinol (12.23 g, 85%) was obtained upon recrys-
1
tallization from toluene; mp 135-37 °C. H NMR (acetone-d6):
δ 3.59 (2H, s, PhCH2), 4.28 (1H, s, CH2COH), 6.71 (2H, d, J )
8.7, ArH), 6.98 (2H, dd, J o ) 6.6, J m ) 2.4, ArH), 7.04-7.07
(3H, m, ArH), 7.13 (1H, t, J ) 7.2, ArH), 7.22 (2H, t, J ) 7.2,
ArH), 7.28 (2H, d, J ) 8.7, ArH), 7.42 (2H, d, J ) 7.2, ArH),
8.21 (1H, s, ArOH). 13C NMR (acetone-d6): δ 48.52 (CH2), 78.18
Mass spectral characterization of synthetic intermediates was
obtained on either a VG Trio 2000 spectrometer (Thermo Onix,

360 Chem. Res. Toxicol., Vol. 16, No. 3, 2003
Gamboa da Costa et al.
(CH2COH), 115.14 (ArCH), 126.49 (ArCH), 126.85 (ArCH),
127.23 (ArCH), 127.90 (ArCH), 128.25 (ArCH), 128.47 (ArCH),
131.92 (ArCH), 138.47 (Cquat), 139.88 (Cquat), 149.06 (Cquat),
156.70 (Cquat). MS (FAB): m/z 290 (M+, 4%), 289 [(M - H)+,
11%], 273 [(MH - H2O)+, 100%], 199 [(MH - C7H8)+, 60%].
Anal. calcd for C20H18O2: C, 82.73%; H, 6.25%. Found: C,
82.83%; H, 6.31%.
methanol (10 mL), and the mixture was stirred at room
temperature for 48 h. Following evaporation of the methanol,
the residue was resuspended in methylene chloride (200 mL)
and washed with water (3 × 0.5 vol). The organic extract was
dried with anhydrous sodium sulfate, filtered, evaporated,
washed three times with n-hexane, and finally dried under
vacuum to yield 10 (644 mg, 86%) as mixture of the E (33%)
and Z (66%) isomers. 1H NMR (DMSO-d6): δ 3.70 (2H, t, J )
3.9, CH2N, E isomer), 3.80 (2H, t, J ) 3.3, CH2N, Z isomer),
4.33 (2H, bs, CH2O, E isomer), 4.48 (2H, bs, CH2O, Z isomer),
6.74 (2H, d, J ) 8.7, alkOPhH, E isomer), 6.89 (2H, d, J ) 8.7,
alkOPhH, E isomer), 6.93-7.40 (10H, E isomer + 14H, Z isomer,
m, ArH). 13C NMR (DMSO-d6): δ 52.09 (m, CD3N), 61.43
(CH2N), 61.57 (CH2N), 63.89 (CH2O), 63.90 (CH2O), 114.16
(ArCH), 114.40 (ArCH), 120.73 (Cquat), 121.11 (Cquat), 127.08
(ArCH), 127.56 (ArCH), 127.98 (ArCH), 128.11 (ArCH), 128.24
(ArCH), 128.35 (ArCH), 128.87 (ArCH), 129.72 (ArCH), 129.84
(ArCH), 130.42 (ArCH), 131.05 (ArCH), 133.51 (Cquat), 136.30
(Cquat), 140.65 (Cquat), 140.81 (Cquat), 143.05 (Cquat), 143.50 (Cquat),
156.26 (Cquat), 156.68 (Cquat). MS (ES): m/z 447 (M+, 100%), 445
(M+, 91%), 367 [(MH - Br)+, 81%].
(E,Z)-1-Br om o-2-[(4-h yd r oxy)p h en yl]-1,2-d ip h en ylet h -
en e (8). A solution of bromine (2.35 mL, 45.6 mmol) in carbon
tetrachloride (15 mL) was added to a solution of 7 (13 g, 44.8
mmol) in the same solvent (100 mL), and the mixture was
stirred at room temperature. The reaction product started to
precipitate after approximately 5 min. The mixture was stirred
for an additional hour and then concentrated to approximately
50% of the original volume. Methylene chloride (150 mL) was
added to dissolve the precipitate, and the solution was washed
sequentially with 1 vol of saturated NaHCO3 and 1 vol of water.
The organic extract was dried with anhydrous sodium sulfate
and evaporated. The bromoalkene 8 (15.22 g, 97%), obtained
as
a mixture of the E (75%) and Z (25%) isomers, was
recrystallized from toluene/n-hexane (3/7). 1H NMR (acetone-
d6): δ 6.57 (2H, d, J ) 8.7, alkOPhH, E isomer), 6.80 (2H, d,
J ) 8.7, alkOPhH, E isomer), 6.86 (2H, d, J ) 8.4, alkOPhH, Z
isomer), 6.96-7.42 (10H, E isomer + 12H, Z isomer, m, ArH),
(E,Z)-1-Br om o-2-[4-[2-(b is(t r id eu t er iom et h yl)a m in o)-
eth oxy]p h en yl]-1,2-d ip h en yleth en e (11). The iodide salt 10
(400 mg, 0.70 mmol) was dissolved in 2-butanone (15 mL) that
had been dried over anhydrous sodium sulfate. Sodium ben-
zenethiolate (470 mg, 3.4 mmol) was then added, and the
mixture was refluxed for 1 h. After the solvent was evaporated,
the residue was resuspended in methylene chloride (100 mL)
and washed sequentially with 0.5 M sodium hydroxide (2 × 1
vol) and water (2 × 1 vol). The organic extract was concentrated,
and the product was purified by flash chromatography. Elution
was started with methylene chloride to remove traces of
thiophenol, and then the product (285 mg, 95%) was recovered
with 10% methanol in methylene chloride as a mixture of the
E (26%) and Z (74%) isomers. 1H NMR (acetone-d6): δ 2.58 (2H,
t, J ) 5.9, CH2N, E isomer), 2.68 (2H, t, J ) 5.9, CH2N, Z
isomer), 3.95 (2H, t, J ) 5.9, CH2O, E isomer), 4.10 (2H, t, J )
5.9, CH2O, Z isomer), 6.65 (2H, d, J ) 8.7, alkOPhH, E isomer),
6.89 (2H, d, J ) 8.7, alkOPhH, E isomer), 6.94-7.39 (10H, E
isomer + 14H, Z isomer, m, ArH). 13C NMR (acetone-d6): δ 45.13
(m, CD3N), 58.68 (CH2N), 58.72 (CH2N), 66.96 (CH2O), 67.10
(CH2O), 114.59 (ArCH), 114.77 (ArCH), 121.47 (Cquat), 121.91
(Cquat), 127.73 (ArCH), 128.22 (ArCH), 128.62 (ArCH), 128.75
(ArCH), 128.84 (Cquat), 128.95 (ArCH), 130.06 (ArCH), 130.96
(ArCH), 131.49 (ArCH), 132.18 (ArCH), 133.97 (Cquat), 136.75
(Cquat), 142.21 (Cquat), 144.42 (Cquat), 144.88 (Cquat), 158.81 (Cquat),
159.25 (Cquat). MS (ES): m/z 430 (MH+, 89%), 428 (MH+, 100%),
350 [(MH2 - Br)+, 77%]. HRMS calcd for C24H18D6BrNO,
427.1412; found, 427.1412.
8.42 (1H, s, ArOH, E isomer), 8.56 (1H, s, ArOH, Z isomer). 13
C
NMR (acetone-d6): δ 115.48 (ArCH), 115.57 (ArCH), 121.08
(Cquat), 122.71 (ArCH), 128.20 (ArCH), 128.60 (ArCH), 128.84
(ArCH), 128.95 (ArCH), 130.10 (ArCH), 131.02 (ArCH), 131.62
(ArCH), 132.32 (ArCH), 132.97 (Cquat), 142.38 (Cquat), 144.64
(Cquat), 145.02 (Cquat), 157.25 (Cquat), 157.38 (Cquat). MS (EI): m/z
352 (M+, 43%), 350 (M+, 41%), 271 [(M - Br)+, 100%]. Anal.
calcd for C20H15BrO: C, 68.39%; H, 4.30%. Found: C, 68.27%;
H, 4.31%.
(E,Z)-1-Br om o-2-[4-(2-a m in oeth oxy)p h en yl]-1,2-d ip h en -
yleth en e (9). To a solution of 8 (8 g, 22.8 mmol) in dioxane/
toluene (1/3, 150 mL) was added a 10-fold molar excess of
powdered potassium hydroxide. The mixture was then taken
to reflux, and an excess of solid 2-chloroethylamine hydrochlo-
ride (ca. 5 equiv) was added portionwise until TLC (silica gel,
15% methanol in methylene chloride) indicated no further
change. The mixture was then concentrated to approximately
50% of the original volume, water (400 mL) was added, and the
organic materials were extracted with methylene chloride (2 ×
0.5 vol). The organic extract was washed sequentially with 0.5
M sodium hydroxide (1 vol) and water (2 × 1 vol), dried with
anhydrous sodium sulfate, and evaporated. The residue was
purified by flash chromatography on silica gel H (Type 60, E.
Merck, Darmstadt, Germany) by eluting with a stepwise gradi-
ent of 0-10% methanol in methylene chloride. The alkylated
product 9 (6.26 g, 70%) was obtained as a mixture of the E (33%)
(E)-4-[4-[2-(Bis(tr ideu ter iom eth yl)am in o)eth oxy]ph en yl]-
3,4-d ip h en ylbu t-3-en -2-ol (12). The synthetic procedure was
based on the method of Foster et al. (67). Briefly, a 120 mM
solution of the (E,Z)-bromoalkene 11 in freshly distilled dry THF
was cooled to -110 °C and kept under argon. A 3-fold molar
excess of 1.6 M n-butyllithium was then added, while keeping
the temperature below -80 °C. The dark brown vinyllithium
intermediate was then quenched by dropwise addition of an
excess of dry acetaldehyde until the mixture became pale yellow.
The excess of acetaldehyde was quickly removed by evaporation,
and saturated aqueous ammonium chloride was added to the
mixture. Following extraction with methylene chloride, 12 was
separated from the Z isomer by TLC on silica gel (Merck) by
eluting twice with methylene chloride/methanol (9/1) and
recovered in 34% yield by precipitation from diethyl ether; mp
121-122 °C. 1H NMR (CDCl3): 1.20 (3H, d, J 6.6, CH3CH), 2.66
(2H, t, J ) 5.9, CH2N), 3.92 (2H, t, J ) 5.9, CH2O), 4.83 (1H, q,
J ) 6.6, CH3CH), 6.55 (2H, d, J ) 8.7, alkOPhH), 6.80 (2H, d,
1
and Z (66%) isomers. H NMR (acetone-d6): δ 3.47 (2H, t, J )
6.0, CH2N, E isomer), 3.57 (2H, t, J ) 6.0, CH2N, Z isomer),
4.06 (2H, t, J ) 6.0, CH2O, E isomer), 4.21 (2H, t, J ) 6.0, CH2O,
Z isomer), 6.66 (2H, d, J ) 8.7, alkOPhH, E isomer), 6.89 (2H,
d, J ) 8.7, alkOPhH, E isomer), 6.95-7.40 (10H, E isomer +
14H, Z isomer, m, ArH). 13C NMR (acetone-d6): δ 51.08 (CH2N),
51.20 (CH2N), 68.86 (CH2O), 69.02 (CH2O), 114.58 (ArCH),
114.76 (ArCH), 121.41 (Cquat), 121.88 (Cquat), 127.72 (ArCH),
128.21 (ArCH), 128.60 (ArCH), 128.73 (ArCH), 128.83 (Cquat),
128.94 (ArCH), 130.05 (ArCH), 130.95 (ArCH), 131.47 (ArCH),
132.16 (ArCH), 133.89 (Cquat), 136.66 (Cquat), 142.18 (Cquat),
144.40 (Cquat), 144.84 (Cquat), 158.91 (Cquat), 159.36 (Cquat), 168.52
(Cquat). MS (EI): m/z 395 (M+, 4%), 393 (M+, 4%), 352 [(M -
CH2CHNH2)+, 7%], 350 [(M - CH2CHNH2)+, 6%], 315 [(M -
Br + 1)+, 35%], 272 [(M - CH2CHNH2 - Br + 1)+, 100%], 178
{[(C6H5)2C2]+, 21%}, 44 [(CH2CH2NH2)+, 14%]. Anal. calcd for
C22H20BrNO: C, 67.01%; H, 5.11%; N, 3.55%. Found: C, 66.82%;
H, 5.14%; N, 3.41%.
J
) 8.7, alkOPhH), 7.17-7.40 (10H, m, PhH). 13C NMR
(E,Z)-1-Br om o-2-[4-[2-(t r is(t r id eu t er iom et h yl)a m m o-
n io)eth oxy]p h en yl]-1,2-d ip h en yleth en e iod id e (10). Potas-
sium carbonate (6.25 g, 45.3 mmol) and iodomethane-d3 (1.4 mL,
22.4 mmol) were added to a solution of 9 (500 mg, 1.3 mmol) in
(CDCl3): 22.46 (CH3CH), 42.69 (m, CD3N), 58.00 (CH2N), 65.49
(CH2O), 68.12 (CH3CH), 113.35 (ArCH), 126.55 (ArCH), 126.99
(ArCH), 127.75 (ArCH), 128.26 (ArCH), 129.51 (ArCH), 131.06
(ArCH), 131.37 (ArCH), 134.54 (Cquat), 138.34 (Cquat), 140.60

HPLC-ES-MS/ MS Quantification of Tamoxifen DNA Adducts
Chem. Res. Toxicol., Vol. 16, No. 3, 2003 361
(Cquat), 141.50 (Cquat), 141.94 (Cquat), 156.93 (Cquat). MS (EI): m/z
393 (M+), 78 [CH2CH2N(CD3)2]+, 64 [CH2N(CD3)2]+. HRMS calcd
for C26H23D6NO2, 393.2569; found, 393.2575.
The adduct levels in each sample were determined by compari-
son of the adduct peak areas to those obtained from injection of
known amounts of the dG-Tam adduct standard.
HP LC-ES-MS/MS An a lyses. (1) LC. The liquid handling
system consisted of an Alliance 2790 pump (Waters Associates),
a Dionex GP40 quaternary gradient pump (Dionex, Sunnyvale,
CA), and two automated switching valves (TPMV, Rheodyne,
Cotati, CA). Valve 1 allowed the pump eluent to either load a
sample onto the trap column and then wash it or to bypass the
trap column and clean the analytical column. Valve 2 was used
to divert the trap column effluent to either waste or the
analytical column. The Alliance 2790 pump was used for sample
injection and cleanup and regeneration of the trap and analyti-
cal columns; the Dionex pump, containing 73% of 0.1% (v/v)
aqueous formic acid and 27% of acetonitrile, was used to
backflush the trap column to the analytical column during
analysis and to keep a constant flow of mobile phase going into
the mass spectrometer during the sample loading and prepara-
tion periods.
Each sample was loaded onto a reversed phase trap column
[Luna C18(2), 2 mm × 30 mm, 3 µm, Phenomenex, Torrance,
CA] and washed to waste for 4.5 min with 95% of 0.1% formic
acid and 5% of acetonitrile at a flow rate of 0.75 mL/min. After
valve 2 was switched, the concentrated sample zone was back-
flushed from the trap column onto the analytical column [Luna
C18(2), 2 mm × 150 mm, 3 µm, Phenomenex] with 73% of 0.1%
formic acid and 27% of acetonitrile at 0.2 mL/min, and the
sample components were eluted into the mass spectrometer.
Following an 8-min run, valve 2 was switched and the trap
column was cleaned to waste with 95% of acetonitrile and 5%
of 0.1% formic acid for 2 min, at a flow rate of 0.75 mL/min.
Next, valve 1 was switched and the analytical column was
cleaned for 2 min with the same eluent, at a flow rate of 0.2
mL/min. Both valves were then switched to their initial posi-
tions, to equilibrate both the trap and the analytical column at
the starting mobile phase compositions, and the process was
repeated.
(2) MS. A Quattro Ultima triple quadrupole mass spectrom-
eter (Micromass, Manchester, U.K.), equipped with an ES
interface, was used with a source block of 150 °C and a
desolvation temperature of 450 °C. Nitrogen was used as the
desolvation (750 L/h) and nebulizing (90 L/h) gas. Argon was
used as the collision gas, at a collision cell pressure of 1.5 ×
10-3 mBar. Positive ions were acquired in the MRM mode (dwell
time of 0.3 s, span of 0.02 Da, and interchannel delay of 0.03 s)
for the transitions from the doubly charged protonated nucleo-
side molecule [(M + 2H)2+] to the doubly charged protonated
purine base [(BH + 2H)2+] for both dG-Tam (m/z 319 f 261)
and dG-desMeTam (m/z 312 f 254), and the internal standard,
dG-Tam-d6 (m/z 322 f 264). The cone voltage was 15 V, and
the collision energy was 9 eV for all three transitions.
DNA Ad d u ct Sta n d a r d s. R-Acetoxytamoxifen was reacted
with DNA using the method of Osborne et al. (36), as modified
by Gamboa da Costa et al. (29). Following sequential extraction
of the unbound materials with diethyl ether and n-butanol, both
of which had been presaturated with 5 mM Bis-Tris and 0.1
mM EDTA (pH 7.1), the DNA was precipitated with sodium
chloride and ethanol and redissolved in 5 mM Bis-Tris and 0.1
mM EDTA (pH 7.1) at a concentration of ∼1 mg/mL. After
enzymatic hydrolysis to nucleosides (68), the adducts were
partitioned into n-butanol, which had been presaturated with
5 mM Bis-Tris and 0.1 mM EDTA (pH 7.1), the n-butanol was
evaporated, and the residue was redissolved in methanol. The
major adduct, dG-Tam (36), was then isolated by HPLC, at a
flow rate of 2 mL/min, using a 17 min linear gradient of 0-60%
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 adduct was thoroughly
dried under vacuum and quantified on the basis of its molar
extinction coefficients (ꢀ250 ) 16 800 M-1 cm-1; ꢀ275 ) 13 200
M-1 cm-1) as described in Gamboa da Costa et al. (29).
dG-desMeTam, the major adduct from reaction of R-sulfoxy-
N-desmethyltamoxifen with DNA, was prepared and purified
as detailed in Gamboa da Costa et al. (28). The deuterated
adduct standard, dG-Tam-d6, was synthesized from 12, isolated
as described above for dG-Tam (29, 36), and characterized
by MS/MS. MS (ES): m/z 643 (MH+). MS/MS (643): m/z 527
[(MH2 - dR)+], 376 [(M - dG)+], 350 [(MH2 - dG - CH3CH)+],
178 [(Gua + CH3CH)+], 78 [CH2CH2N(CD3)2]+.
For quantification purposes, the molar extinction coefficients
of both dG-desMeTam and dG-Tam-d6 were assumed to be
identical to those determined for dG-Tam, since the minor
structural differences between the three adducts were not
expected to affect the corresponding chromophores. Stock solu-
tions containing known concentrations of dG-Tam, dG-des-
MeTam, or dG-Tam-d6 in methanol were prepared and kept at
-20 °C in vials sealed with Teflon septa to prevent evaporation
of the solvent.
In Vitr o Mod ified DNA Sa m p les. DNA samples with
different extents of modification were prepared by conducting
a series of overnight incubations at 37 °C in which R-acetoxy-
tamoxifen was reacted with salmon testis DNA at ratios varying
by 10-fold increments, from 0.1 ng to 1 mg R-acetoxytamoxifen/
mg DNA. After it was purified, as described above, the DNA
was redissolved in 5 mM Bis-Tris and 0.1 mM EDTA (pH 7.1)
at a concentration of ∼1.7 mg/mL. For comparative purposes, a
control incubation was conducted without R-acetoxytamoxifen.
Aliquots of each solution, containing 100 µg of DNA, were
subsequently hydrolyzed to nucleosides (68) and analyzed
directly by either HPLC-UV or HPLC-ES-MS/MS without prior
partition of the adducts into n-butanol.
Resu lts
Tr ea tm en t of An im a ls. Four female Sprague-Dawley rats
(8 weeks old, obtained from the breeding colony at the National
Center for Toxicological Research) were treated by gavage (29)
with seven daily doses of tamoxifen (20 mg/kg, 54 µmol/kg,
dissolved in 200 µL of trioctanoin). Four additional control rats
were treated in the same manner with 200 µL of trioctanoin
alone. Twenty-four hours after the last treatment, the animals
were killed by exposure to carbon dioxide and the livers and
uteri were quickly excised. Hepatic nuclei were isolated by the
method of Basler et al. (69), and DNA was prepared from the
liver nuclei and uteri by slight modifications of the procedure
described in Beland et al. (70).
HP LC-UV An a lyses. HPLC-UV quantification of in vitro
modified DNA samples was conducted by direct injection of the
DNA hydrolysates. The adducts were separated at a flow rate
of 2 mL/min, using a 20-min linear gradient of 20-60%
acetonitrile in 100 mM ammonium acetate (pH 5.7), followed
by a 5-min isocratic elution with 60% acetonitrile in the same
buffer, and a 10-min linear gradient of 60-100% acetonitrile.
Syn t h esis of t h e Deu t er a t ed Ad d u ct St a n d a r d ,
d G-Ta m -d 6. Adduct quantitation by mass spectrometry
is best achieved by coupling selected ion recording or
MRM with the use of a stable isotope-labeled internal
standard (65). Therefore, a simple synthetic strategy was
devised to obtain a deuterated analogue of dG-Tam.
Deuteration of the two methyl groups attached to the
alkylamino substituent was judged appropriate to provide
sufficient m/z separation from dG-Tam, since no lability
of these groups was expected during the synthetic or
analytical procedures. The approach to obtain the adduct
precursor, 12, is outlined in Figure 3. The preparation
of the bromoalkene 9 followed standard procedures,
involving bromination of the benzylic carbon of 7, with
concomitant dehydration to 8, and then alkylation with
2-chloroethylamine hydrochloride in alkali to yield 9. The
problem of controlled trideuteriomethylation to a tertiary

362 Chem. Res. Toxicol., Vol. 16, No. 3, 2003
Gamboa da Costa et al.
F igu r e 4. MS/MS characterization of the deuterated adduct
standard, dG-Tam-d6, using ES ionization. The major fragments
derived from the protonated molecule (M + H)+ are outlined in
the structure.
amine was circumvented by full alkylation with CD3I to
the ammonium salt 10, followed by removal of one
trideuteriomethyl group through nucleophilic displace-
ment with benzenethiolate (71). The bromoalkene 11 was
then converted to 12 through lithiation and subsequent
addition of acetaldehyde, essentially as described for
R-hydroxytamoxifen (67). Spectroscopic data for 12 (1H
and 13C NMR, standard EI-MS, and HRMS; cf. Materials
and Methods) were in full agreement with the assigned
structure.
F igu r e 5. HPLC-UV analysis of a hydrolyzed DNA sample
from a modification conducted in vitro with 1 mg of R-acetoxy-
tamoxifen/mg DNA (panel B). The absorbance profile obtained
from nonmodified DNA (panel A) is shown for comparison. The
elution conditions are outlined in Materials and Methods.
Determination of the total extent of modification was based upon
the molar extinction coefficient of dG-Tam, as described in
Materials and Methods.
Synthesis of dG-Tam-d6 from 12 was performed as
described for dG-Tam (29, 36). The adduct had an HPLC
retention time and UV spectrum similar to those of its
nondeuterated analogue (not shown). MS, with ES ion-
ization, indicated a protonated molecule (m/z 643) con-
sistent with the expected structure. A product ion analy-
sis of the protonated molecule in the ES-MS/MS mode
further confirmed the characterization of the isolated
adduct as dG-Tam-d6 (Figure 4). Thus, the fragment ions
detected at m/z 527 and 376 corresponded to loss of
deoxyribose and deoxyguanosine, respectively, while a
small fragment ion at m/z 78, consistent with N,N-bis-
(trideuteriomethyl)aziridinium [CH2CH2N(CD3)2+], con-
firmed the incorporation of the trideuteriomethyl groups
in the aminoethoxy substituent. Additional fragment
ions at m/z 350 [(MH2 - dG - CH3CH)+] and m/z 178
[(Gua + CH3CH)+] were fully compatible with attachment
of deoxyguanosine to the allylic carbon of the deuterated
tamoxifen segment. This fragmentation pattern was
virtually identical to that obtained for dG-Tam (not
shown).
In Vitr o Mod ifica tion of Sa lm on Testis DNA w ith
r-Acetoxyta m oxifen . R-Acetoxytamoxifen was prepared
by acetylation of R-hydroxytamoxifen with acetic anhy-
dride and reacted with salmon testis DNA (29, 36) at
different ratios, varying by 10-fold increments from 0.1
ng to 1 mg R-acetoxytamoxifen/mg DNA. After the DNA
was purified by solvent extractions and precipitation,
aliquots of the highest modified samples, containing 100
µg of DNA, were hydrolyzed enzymatically to nucleosides
and the hydrolysates were analyzed directly by HPLC
with UV detection. Figure 5 depicts representative chro-
matograms obtained with nonmodifed DNA (panel A) and
with DNA modified with 1 mg of R-acetoxytamoxifen/mg
DNA (panel B). With the modified DNA, two peaks were
present at a ratio of approximately 4:1. The major peak
co-chromatographed with the synthetic standard, dG-
Ta ble 1. Ad d u ct Levels (Exp r essed a s d G-Ta m /108
Nu cleotid es), a s Deter m in ed by HP LC/UV a n d
HP LC-ES-MS/MS, in DNA Sa m p les Mod ified In Vitr o
w ith r-Acetoxyta m oxifen
reaction ratioa
HPLC/UVb
HPLC-ES-MS/MSc
1
2 070 000
580 000
39 000
NAd
1 520 000
540 000
48 000
5500
560
10-1
10-2
10-3
10-4
10-5
10-6
10-7
NA
NA
NA
NA
61
20
8
a
Expressed as mg of R-acetoxytamoxifen/mg DNA. b The amount
of dG-Tam as determined by comparison of the HPLC peak area
with those of injected dG-Tam standards of known concentration.
c The amount of dG-Tam as determined by HPLC-ES-MS/MS by
d
quantifying against a dG-Tam-d6 internal standard. NA, not
analyzed.
Tam, whereas the minor peak corresponded to a diaste-
reomer or a mixture of diastereomers of the same adduct
(37, 38). The total extent of modification for the sample
represented in Figure 5B was estimated to be 207
adducts/104 nucleotides (Table 1). This estimate was
based upon the molar extinction coefficient determined
for the major adduct, dG-Tam (29), and on the assump-
tion of a similar value for the adduct(s) in the minor peak,
since both epimerization at the R carbon of the tamoxifen
segment and E-Z isomerization of the olefinic double
bond were not expected to introduce significant alter-
ations in the chromophore. A similar adduct pattern was
observed with DNA samples modified with 0.1 and 0.01
mg of R-acetoxytamoxifen/mg DNA, with the adduct
levels being estimated to be 58 and 3.9 adducts/104
nucleotides, respectively (Table 1). The adduct levels in
the samples modified with lower ratios of R-acetoxyta-
moxifen/DNA were too low to be detected by HPLC-UV
when using 100 µg of DNA.

HPLC-ES-MS/ MS Quantification of Tamoxifen DNA Adducts
Chem. Res. Toxicol., Vol. 16, No. 3, 2003 363
F igu r e 6. Representative HPLC-ES-MS/MS analyses of tamoxifen DNA adducts in liver DNA from control (panels A, C, and E)
and tamoxifen-treated female Sprague-Dawley rats (panels B, D, and F). The rats were treated by gavage with seven daily doses
of 20 mg tamoxifen/kg body weight and killed 24 h after the last dose, as outlined in Materials and Methods. Panels A and B: MRM
(relative signal intensity vs time) for the transition from the doubly charged protonated nucleoside molecule [(M + 2H)2+] to the
doubly charged protonated purine base [(BH + 2H)2+] for 100 pg of the internal standard, dG-Tam-d6 (m/z 322 f 264). Panels C and
D: MRM for the transition from [(M + 2H)2+] to [(BH + 2H)2+] for dG-Tam (m/z 319 f 261). Panels E and F: MRM for the transition
from [(M + 2H)2+] to [(BH + 2H)2+] for dG-desMeTam (m/z 312 f 254). The retention time for the most prominent peak in each
chromatogram is indicated. The elution conditions are outlined in Materials and Methods.
HP LC-ES-MS/MS An a lyses of DNA Sa m p les Mod i-
fied In Vitr o w ith r-Acetoxyta m oxifen . The HPLC-
ES-MS/MS method was validated with respect to intra-
and interassay precision and accuracy by analyzing, on
different days, an unmodified salmon testis DNA hy-
drolysate (100 µg) to which dG-Tam (10 pg) was added.
On day 1, the dG-Tam level measured in the sample was
0.103 ( 0.003 pg/µg DNA (2.9% RSD, n ) 4). On day 2,
the dG-Tam level was found to be 0.105 ( 0.003 (3.1%
RSD, n ) 4). This corresponds to an adduct level of 5.2
dG-Tam adducts in 108 normal nucleotides. No observ-
able responses for either dG-Tam or dG-desMeTam were
detected in the control DNA in the absence of added
adduct standards. The LOD (S/N ) 3) was approximately
1 pg on-column, which is equivalent to 5 dG-Tam adducts
in 109 normal nucleotides, and the LOQ (S/N ) 10) was
approximately 1 dG-Tam adduct in 108 normal nucleo-
tides. In the presence of 25 pg of the deuterium-labeled
internal standard (dG-Tam-d6), a plot of the response
ratio vs the concentration ratio (not shown) was found
to be linear from the LOD up to 250 pg of dG-Tam
(correlation coefficient > 0.99; slope, ∼1). Similarly, the
response for dG-desMeTam (not shown) was also deter-
mined to be linear (correlation coefficient > 0.99; slope,
0.085). The method was validated within the range of
8-1 520 000 adducts/108 nucleotides, using 100 µg of the
various DNA samples modified in vitro to different
extents with R-acetoxytamoxifen. Although the process
produced no observable analyte carryover to the subse-
quent injection, injections of reagent blanks, control DNA
hydrolysates, and adduct standards were regularly in-
corporated into analytical runs for confirmation.
R-acetoxytamoxifen/mg DNA. The correspondence be-
tween the results from the two methods is very good; in
addition, the regular variation in adduct levels obtained
for the different samples is consistent with what would
be expected from their corresponding reaction ratios.
HP LC-ES-MS/MS An a lyses of DNA Mod ified In
Vivo w ith Ta m oxifen . Female Sprague-Dawley rats
were treated by gavage (29) with seven daily doses of
tamoxifen (20 mg/kg). Hepatic and uterine DNA was
isolated, and the extent of binding was assessed by
HPLC-ES-MS/MS using conditions identical to those
described for analysis of the in vitro modified DNA.
Figure 6 illustrates representative HPLC-ES-MS/MS
analyses of liver DNA from a control rat (panels A, C,
and E) and from a rat administered tamoxifen (panels
B, D, and F). The traces corresponding to MRM of the
transitions from the doubly charged protonated nucleo-
side molecule [(M + 2H)2+] to the doubly charged pro-
tonated purine base [(BH + 2H)2+] for the internal
standard, dG-Tam-d6 (m/z 322 f 264), are presented in
panels A and B. The corresponding MRM traces for dG-
Tam (m/z 319 f 261) and dG-desMeTam (m/z 312f 254)
are presented in panels C and D and panels E and F,
respectively. As expected, the signal from the deuterated
internal standard is clearly present in both samples,
whereas the signals ascribed to dG-Tam and dG-des-
MeTam were readily seen in the livers from the tamox-
ifen-treated animals but not in the hydrolysates from
control livers. The identity of the signals at 5.98 and 5.94
min, which were detected at high expansion in the m/z
319 f 261 and 312f 254 MRM traces of the control liver
DNA samples (Figure 6), is not known. The dG-Tam and
dG-desMeTam adduct levels determined by HPLC-ES-
MS/MS in the liver DNA from tamoxifen-treated rats
were 496 ( 16 and 626 ( 18 adducts/108 nucleotides,
respectively. Neither dG-Tam nor dG-desMeTam was
detected in the uterus DNA, with the LOD being 5
Table 1 outlines the adduct levels determined by
HPLC-ES-MS/MS for the DNA samples modified in vitro
at ratios of 1-10-7 mg of R-acetoxytamoxifen/mg DNA.
The table also presents the values determined by HPLC-
UV for the samples modified at ratios of 1-10-2 mg of

364 Chem. Res. Toxicol., Vol. 16, No. 3, 2003
Gamboa da Costa et al.
adducts/109 nucleotides for dG-Tam and 5 adducts/108
nucleotides for dG-desMeTam.
advantage of ES-MS was further enhanced by the use of
MS/MS, which can provide information on specific frag-
mentation patterns, and MRM, which results in very low
analytical backgrounds (65).
Discu ssion
In the present study, we have explored the HPLC-ES-
MS/MS methodology as a suitable routine analytical tool
for the detection and quantification of tamoxifen DNA
adducts formed in vivo. The method involved on-line
sample preparation and cleanup and MRM of the transi-
tions from the doubly charged protonated nucleoside
molecule [(M + 2H)2+] to the doubly charged protonated
purine base [(BH + 2H)2+] for dG-Tam, dG-desMeTam,
and an internal standard, dG-Tam-d6. By quantifying
against the deuterated internal standard, the method
was validated within the range of 8-1 520 000 adducts/
108 nucleotides using 100 µg of hydrolyzed DNA that had
been modified in vitro with R-acetoxytamoxifen. The LOD
and LOQ levels for dG-Tam were estimated at 5 adducts/
109 nucleotides and 1 adduct/108 nucleotides, respectively,
and good statistical performance (RSD ∼3%) was ob-
tained. In addition, dG-Tam and dG-desMeTam were
readily detected in rat liver DNA of female Sprague-
Dawley rats administered tamoxifen under a typical
dosing regimen (29). DNA adducts were not detected in
uterus DNA, which confirms results previously obtained
by 32P-postlabeling analyses (29).
In conclusion, we have shown that HPLC-ES-MS/MS
has sufficient sensitivity and precision to be useful in the
routine analysis of tamoxifen DNA adducts formed in
vivo in experimental animals. Despite being somewhat
less sensitive than the 32P-postlabeling or immunoassay
methodologies, it has the clear advantage of providing
information on the chemical identities of the adducts.
Some investigators have reported tamoxifen DNA ad-
ducts to be present at 0.2-18 adducts/108 nucleotides in
endometrial samples from women undergoing tamoxifen
therapy (59, 60). In preliminary studies, we have used
HPLC-ES-MS/MS to assess the presence of dG-Tam in
endometrial DNA from women treated daily with 20 mg
of tamoxifen for up to 5 years. We did not detect dG-Tam
in any of these samples (56 and unpublished data), which
is similar to what has been reported by other investiga-
tors using 32P-postlabeling techniques (61, 62). Additional
studies are clearly necessary to resolve this controversy;
nonetheless, the HPLC-ES-MS/MS methodology de-
scribed in this paper should help clarify the genotoxic
potential of tamoxifen in the human endometrium.
Tamoxifen has been associated with an increased
incidence of endometrial cancer, when used both as a
chemotherapeutic or as a chemopreventive agent against
breast cancer (1, 2). Although the mechanism for the
induction of these tumors is not known, conflicting
evidence regarding the detection of DNA adducts in
endometrial samples from women treated with tamoxifen
(57-63) has led to suggestions that the estrogen agonist
effect of tamoxifen, promoting uterine cell proliferation,
may be involved (72). Tamoxifen and some of its metabo-
lites, such as N-desmethyltamoxifen (2), N,N-didesmeth-
yltamoxifen, and 4-hydroxytamoxifen, have been detected
in endometrial samples from women on tamoxifen therapy
at estimated levels of approximately 2-4 µg/g of tissue
(73). In addition, cytochrome P450 enzymes, including
3A isoforms that have been implicated in the activation
of tamoxifen to R-hydroxytamoxifen (31), appear to be
expressed in the human endometrium (74). Therefore,
the possibility of tamoxifen being activated in the human
uterus by R-hydroxylation seems plausible, and even in
the absence of significant sulfotransferase activity to
ensure further activation (5), the intrinsic reactivity of
R-hydroxytamoxifen (and presumably R-hydroxy-N-des-
methyltamoxifen (4)) toward DNA at physiological pH
(36) could conceivably generate uterine DNA adducts.
Thus, to establish whether tamoxifen acts as a genotoxic
agent in the human endometrium, the use of reliable
adduct detection methodologies to search for putative
tamoxifen DNA adducts is of critical importance, not only
to establish if adducts are present but also to establish
if the detected adduct levels are biologically relevant.
32P-postlabeling has been the most widely used meth-
odology for the assessment of tamoxifen DNA adducts,
formed both in vitro and in vivo, with LODs reported in
the ranges of 1 adduct/109 nucleotides (62) to approxi-
mately 2.5 adducts/1010 nucleotides (59, 60). More re-
cently, competitive DELFIA and CIA have been used to
quantify tamoxifen DNA adducts (64), with the latter
technique having a LOD estimated at 3 adducts/109
nucleotides. Despite the high sensitivity achieved in
adduct detection by both the 32P-postlabeling and immu-
noassay methodologies, none of these techniques can
provide a chemical characterization of the adducts (65).
Furthermore, quantitation by 32P-postlabeling can suffer
from a series of limitations including incomplete DNA
digestion, inefficiency of adduct labeling by T4 polynu-
cleotide kinase, and loss of adducts during the enrich-
ment process (65), whereas immunoassay results may,
for instance, be limited by crossreactivity of the antisera.
MS is a technique of great potential when applied to
DNA adduct detection, due to its high chemical specific-
ity, which permits the identification of particular DNA
adducts even in the presence of other species, including
chemically unrelated adducts. We have previously used
HPLC-ES-MS in the analysis of DNA samples modified
in vitro and in vivo with the arylamine carcinogen
4-aminobiphenyl (75, 76). More recently, HPLC-ES-MS/
MS, using MRM, was applied with good results to the
routine quantification of etheno DNA adducts formed in
vitro, and also in animal tissues and human placenta
(77), at levels of 1 adduct in 108 nucleotides. The intrinsic
Ack n ow led gm en t. We thank Indale´cio Marques for
the FAB mass spectra, J oaquim Marc¸alo for the high-
resolution mass spectra, V´ıtor Pereira for some of the
elemental analyses, and R. Wayne Benson for assisting
in the collection of animal tissues. This work was
supported in part by a Postgraduate Research Program
administered by the Oak Ridge Institute for Science and
Education, by research grants from Programs PRAXIS
XXI and POCTI, Fundac¸a˜o para a Cieˆncia e a Tecnologia
(FCT), Portugal, and by a fellowship to G.G.C. from
Subprograma Cieˆncia e Tecnologia, FCT, Portugal.
Refer en ces
(1) Early Breast Cancer Trialists’ Collaborative Group (1998) Tamox-
ifen for early breast cancer: an overview of the randomised trials.
Lancet 351, 1451-1467.
(2) Fisher, B., Costantino, J . P., Wickerham, D. L., Redmond, C. K.,
Kavanah, M., Cronin, W. M., Vogel, V., Robidoux, A., Dimitrov,
N., Atkins, J ., Daly, M., Wieand, S., Tan-Chiu, E., Ford, L.,

HPLC-ES-MS/ MS Quantification of Tamoxifen DNA Adducts
Chem. Res. Toxicol., Vol. 16, No. 3, 2003 365
Wolmark, N., and other National Surgical Adjuvant Breast and
Bowel Project Investigators (1998) Tamoxifen for prevention of
breast cancer: Report of the National Surgical Adjuvant Breast
and Bowel Project P-1 Study. J . Natl. Cancer Inst. 90, 1371-
1388.
(23) Li, D., Dragan, Y., J ordan, V. C., Wang, M., and Pitot, H. C. (1997)
Effects of chronic administration of tamoxifen and toremifene on
DNA adducts in rat liver, kidney, and uterus. Cancer Res. 57,
1438-1441.
(24) Brown, K., Brown, J . E., Martin, E. A., Smith, L. L., and White,
I. N. H. (1998) Determination of DNA damage in F344 rats
induced by geometric isomers of tamoxifen and analogues. Chem.
Res. Toxicol. 11, 527-534.
(25) Martin, E. A., Heydon, R. T., Brown, K., Brown, J . E., 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.
(26) 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 metabolic activation of
tamoxifen in rat liver cells. Carcinogenesis 20, 2003-2009.
(27) 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.
(28) 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.
(29) Gamboa da Costa, G., McDaniel-Hamilton, L. P., Heflich, R. H.,
Marques, M. M., and Beland, F. A. (2001) DNA adduct formation
and mutant induction in Sprague-Dawley rats treated with
tamoxifen and its derivatives. Carcinogenesis 22, 1307-1315.
(30) White, I. N. H., Carthew, P., Davies, R., Styles, J ., Brown, K.,
Brown, J . E., Smith, L. L., and Martin, E. A. (2001) Short-term
dosing of R-hydroxytamoxifen results in DNA damage but does
not lead to liver tumours in female Wistar/Han rats. Carcino-
genesis 22, 553-557.
(3) Bergman, L., Beelen, M. L. R., Gallee, M. P. W., Hollema, H.,
Benraadt, J ., van Leeuwen, F. E., and the Comprehensive Cancer
Centres’ ALERT Group. (2000) Risk and prognosis of endometrial
cancer after tamoxifen for breast cancer. Lancet 356, 881-887.
(4) Deligdisch, L., Kalir, T., Cohen, C. J ., de Latour, M., Le Bouedec,
G., and Penault-Llorca, F. (2000) Endometrial histopathology in
700 patients treated with tamoxifen for breast cancer. Gynecol.
Oncol. 78, 181-186.
(5) Phillips, D. H. (2001) Understanding the genotoxicity of tamox-
ifen? Carcinogenesis 22, 839-849.
(6) Webb, P., Lopez, G. N., Uht, R. M., and Kushner, P. J . (1995)
Tamoxifen activation of the estrogen receptor/AP-1 pathway:
potential origin for the cell-specific estrogen-like effects of anti-
estrogens. Mol. Endocrinol. 9, 443-456.
(7) Branham, W. S., Fishman, R., Streck, R. D., Medlock, K. L.,
DeGeorge, J . J ., and Sheehan, D. M. (1996) ICI 182, 780 inhibits
endogenous estrogen-dependent rat uterine growth and tamox-
ifen-induced developmental toxicity. Biol. Reprod. 54, 160-167.
(8) Schwartz, L. B., Krey, L., Demopoulos, R., Goldstein, S. R.,
Nachtigall, L. E., and Mittal, K. (1997) Alterations in steroid
hormone receptors in the tamoxifen-treated endometrium. Am.
J . Obstet. Gynecol. 176, 129-137.
(9) Elkas, J ., Armstrong, A., Pohl, J ., Cuttitta, F., Mart´ınez, A., and
Gray, K. (2000) Modulation of endometrial steroid receptors and
growth regulatory genes by tamoxifen. Obstet. Gynecol. 95, 697-
703.
(10) Karck, U., and Kommoss, F. (2000) Does tamoxifen change
oestrogen and progesterone receptor expression in the endometri-
um and breast? Eur. J . Cancer 36, S45-S46.
(11) 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.
(12) 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 CrI:CD(BR) rats.
Cancer Res. 53, 4534-4541.
(13) 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.
(31) Mani, C., Pearce, R., Parkinson, A., and Kupfer, D. (1994)
Involvement of cytochrome P4503A in catalysis of tamoxifen
activation and covalent binding to rat and human liver mi-
crosomes. Carcinogenesis 15, 2715-2720.
(32) 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.
(33) Glatt, H., Davis, W., Meinl, W., Hermesdo¨rfer, H., Venitt, S., and
Phillips, D. H. (1998) Rat, but not human, sulfotransferase
activates a tamoxifen metabolite to produce DNA adducts and
gene mutations in bacteria and mammalian cells in culture.
Carcinogenesis 19, 1709-1713.
(34) Shibutani, S., Shaw, P. M., Suzuki, N., Dasaradhi, L., Duffel, M.
W., and Terashima, I. (1998) Sulfation of R-hydroxytamoxifen
catalyzed by human hydroxysteroid sulfotransferase results in
tamoxifen-DNA adducts. Carcinogenesis 19, 2007-2011.
(35) Glatt, H., Bartsch, I., Cristoph, S., Coughtrie, M. W. H., Falany,
C. N., Hagen, M., Landsiedel, R., Pabel, U., Phillips, D. H., Seidel,
A., and Yamazoe, Y. (1998) Sulfotransferase-mediated activation
of mutagens studied using heterologous expression systems.
Chem. Biol. Interact. 109, 195-219.
(14) Ma¨ntyla¨, E., Nieminen, L., and Karlsson, S. (1995) Endometrial
cancer induction by tamoxifen in the rat. Eur. J . Cancer 31A
(Suppl. 5), S14.
(15) Ma¨ntyla¨, E. T. E., Karlsson, S. H., and Nieminen, L. S. (1996)
Induction of endometrial cancer by tamoxifen in the rat. in
Hormonal Carcinogenesis. II. Proceedings of the Second Interna-
tional Symposium (Li, J J ., Li, S. A., Gustafsson, J .-Å., Nandi, S.,
and Sekely, L. I., Eds.) pp 442-445, New York, Springer-Verlag.
(16) Newbold, R. R., J efferson, W. N., Padilla-Burgos, E., and Bullock,
B. C. (1997) Uterine carcinoma in mice treated neonatally with
Tamoxifen. Carcinogenesis 18, 2293-2298.
(36) 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.
(37) 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.
(17) Diwan, B. A., Anderson, L. M., and Ward, J . M. (1997) Prolifera-
tive lesions of oviduct and uterus in CD-1 mice exposed prenatally
to tamoxifen. Carcinogenesis 18, 2009-2014.
(18) Carthew, P., Edwards, R. E., Nolan, B. M., Martin, E. A., Heydon,
R. T., White, I. N. H., and Tucker, M. J . (2000) Tamoxifen induces
endometrial and vaginal cancer in rats in the absence of endome-
trial hyperplasia. Carcinogenesis 21, 793-797.
(38) 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.
(39) Rajaniemi, H., Koskinen, M., Ma¨ntyla¨, E., and Hemminki, K.
(1998) DNA binding of tamoxifen and its analogues: identification
of the tamoxifen-DNA adducts in rat liver. Toxicol. Lett. 102-
103, 453-457.
(19) Han, X., and Liehr, J . G. (1992) Induction of covalent DNA
adducts in rodents by tamoxifen. Cancer Res. 52, 1360-1363.
(20) 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 C57BL/6 mice and in human MCL-5 cells.
Carcinogenesis 13, 2197-2203.
(40) Rajaniemi, H., Rasanen, I., Koivisto, P., Peltonen, K., and
Hemminki, K. (1999) Identification of the major tamoxifen-DNA
adducts in rat liver by mass spectroscopy. Carcinogenesis 20,
305-309.
(41) Kitagawa, M., Ravindernath, A., Suzuki, N., Rieger, R., Terash-
ima, I., Umemoto, A., and Shibutani, S. (2000) Identification of
tamoxifen-DNA adducts induced by R-acetoxy-N-desmethylta-
moxifen. Chem. Res. Toxicol. 13, 761-769.
(21) Randerath, K., Bi, J ., Mabon, N., Sriram, P., and Moorthy, B.
(1994) Strong intensification of mouse hepatic tamoxifen DNA
adduct formation by pretreatment with the sulfotransferase
inhibitor and ubiquitous environmental pollutant pentachlo-
rophenol. Carcinogenesis 15, 797-800.
(22) Carthew, P., Rich, K. J ., Martin, E. A., de Matteis, F., Lim, C.-
K., Manson, M. M., Festing, M. F. W., White, I. N. H., and Smith,
L. L. (1995) DNA damage as assessed by 32P-postlabelling in three
rat strains exposed to dietary tamoxifen: the relationship between
cell proliferation and liver tumour formation. Carcinogenesis 16,
1299-1304.
(42) Umemoto, A., Monden, Y., Suwa, M., Kanno, Y., Suzuki, M., Lin,
C.-X., Ueyama, Y., Momen, Md. A., Ravindernath, A., Shibutani,
S., and Komaki, K. (2000) Identification of hepatic tamoxifen-

366 Chem. Res. Toxicol., Vol. 16, No. 3, 2003
Gamboa da Costa et al.
DNA adducts in mice: R-(N2-deoxyguanosinyl)tamoxifen and
R-(N2-deoxyguanosinyl)tamoxifen N-oxide. Carcinogenesis 21,
1737-1744.
(60) Shibutani, S., Ravindernath, A., Suzuki, N., Terashima, I.,
Sugarman, S. M., Grollman, A. P., and Pearl, M. L. (2000)
Identification of tamoxifen-DNA adducts in the endometrium of
women treated with tamoxifen. Carcinogenesis 21, 1461-1467.
(61) 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.
(62) Carmichael, P. L., Sardar, S., Crooks, N., Neven, P., Van Hoof,
I., Ugwumadu, A., Bourne, T., Tomas, E., Hellberg, P., Hewer,
A. J ., and Phillips, D. H. (1999) Lack of evidence from HPLC 32P-
post-labelling for tamoxifen-DNA adducts in the human en-
dometrium. Carcinogenesis 20, 339-342.
(63) Martin, E. A., Gaskell, M., White, I. N. H., Garner, R. C., Cupid,
B., Dingley, K., Mauthe, R., Turteltaub, K., Mattock, E., Pring,
D. W., and Smith, L. L. (1998) Analysis of tamoxifen-DNA adducts
in humans and rats by accelerator mass spectrometry. Mutagen-
esis 13, 528.
(64) Divi, R. L., Osborne, M. R., Hewer, A., Phillips, D. H., and Poirier,
M. C. (1999) Tamoxifen-DNA adduct formation in rat liver
determined by immunoassay and 32P-postlabeling. Cancer Res.
59, 4829-4833.
(65) Phillips, D. H., Farmer, P. B., Beland, F. A., Nath, R. G., Poirier,
M. C., Reddy, M. V., and Turteltaub, K. W. (2000) Methods of
DNA adduct determination and their application to testing
compounds for genotoxicity. Environ. Mol. Mutagen. 35, 222-233.
(66) Perrin, D. D., and Armarego, W. L. F. (1988) Purification of
Laboratory Chemicals (3rd ed.) pp 1-391, Pergamon, Oxford, U.K.
(67) 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.
(68) Heflich, R. H., Morris, S. M., Beranek, D. T., McGarrity, L. J .,
Chen, J . J ., and Beland, F. A. (1986) Relationships between the
DNA adducts and the mutations and sister-chromatid exchanges
produced in Chinese hamster ovary cells by N-hydroxy-2-ami-
nofluorene, N-hydroxy-N′-acetylbenzidine and 1-nitrosopyrene.
Mutagenesis 1, 201-206.
(69) Basler, J ., Hastie, N. D., Pietras, D., Matsui, S.-I., Sandberg, A.
A., and Berezney, R. (1981) Hybridization of nuclear matrix
attached deoxyribonucleic acid fragments. Biochemistry 20, 6921-
6929.
(70) Beland, F. A., Fullerton, N. F., and Heflich, R. H. (1984) Rapid
isolation, hydrolysis and chromatography of formaldehyde-modi-
fied DNA. J . Chromatogr. 308, 121-131.
(71) Shamma, M., Deno, N. C., and Remar, J . F. (1966) The selective
demethylation of quaternary ammonium salts. Tetrahedron Lett.
1375-1379.
(72) White, I. N. H. (2001) Anti-oestrogenic drugs and endometrial
cancers. Toxicol. Lett. 120, 21-29.
(73) Giorda, G., Franceschi, L., Crivellari, D., Magri, M. D., Veronesi,
A., Scarabelli, C., and Furlanut, M. (2000) Determination of
tamoxifen and its metabolites in the endometrial tissue of long-
term treated women. Eur. J . Cancer 36, S88-S89.
(74) Hukkanen, J ., Ma¨ntyla¨, M., Kangas, L., Wirta, P., Hakkola, J .,
Paakki, P., Evisalmi, S., Pelkonen, O., and Raunio, H. (1998)
Expression of cytochrome P450 genes encoding enzymes active
in the metabolism of tamoxifen in human uterine endometrium.
Pharmacol. Toxicol. 82, 93-97.
(75) Beland, F. A., Doerge, D. R., Churchwell, M. I., Poirier, M. C.,
Schoket, B., and Marques, M. M. (1999) Synthesis, characteriza-
tion, and quantitation of a 4-aminobiphenyl-DNA adduct stan-
dard. Chem. Res. Toxicol. 12, 68-77.
(76) Doerge, D. R., Churchwell, M. I., Marques, M. M., and Beland,
F. A. (1999) Quantitative analysis of 4-aminobiphenyl-C8-deoxy-
guanosyl DNA adducts produced in vitro and in vivo using HPLC-
ES-MS. Carcinogenesis 20, 1055-1061.
(77) Doerge, D. R., Churchwell, M. I., Fang, J .-L., and Beland, F. A.
(2000) Quantitation of etheno-DNA adducts using liquid chro-
matography, on-line sample processing, and electrospray tandem
mass spectrometry. Chem. Res. Toxicol. 13, 1259-1264.
(43) Umemoto, A., Komaki, K., Monden, Y., Suwa, M., Kanno, Y.,
Kitagawa, M., Suzuki, M., Lin, C.-X., Ueyama, Y., Momen, Md.
A., Ravindernath, A., and Shibutani, S. (2001) Identification and
quantification of tamoxifen-DNA adducts in the liver of rats and
mice. Chem. Res. Toxicol. 14, 1006-1013.
(44) Vancutsem, P. M., Lazarus, P., and Williams, G. M. (1994)
Frequent and specific mutations of the rat p53 gene in hepato-
carcinomas induced by tamoxifen. Cancer Res. 54, 3864-3867.
(45) Davies, R., Oreffo, V. I. C., Bayliss, S., Dinh, P.-A., Lilley, K. S.,
White, I. N. H., Smith, L. L., and Styles, J . A. (1996) Mutational
spectra of tamoxifen-induced mutations in the livers of lacI
transgenic rats. Environ. Mol. Mutagen. 28, 430-433.
(46) Davies, R., Oreffo, V. I. C., Martin, E. A., Festing, M. F. W., White,
I. N. H., Smith, L. L., and Styles, J . A. (1997) Tamoxifen causes
gene mutations in the livers of lambda/lacI transgenic rats.
Cancer Res. 57, 1288-1293.
(47) Davies, R., Gant, T. W., Smith, L. L., and Styles, J . A. (1999)
Tamoxifen induces G:C f T:A mutations in the cII gene in the
liver of lambda/lacI transgenic rats but not at 5′-CpG-3′ dinu-
cleotide sequences as found in the lacI transgene. Carcinogenesis
20, 1351-1356.
(48) Styles, J . A., Davies, R., Fenwick, S., Walker, J ., White, I. N. H.,
and Smith, L. L. (2001) Tamoxifen mutagenesis and carcinogen-
esis in livers of lambda/lacI transgenic rats: selective influence
of phenobarbital promotion. Cancer Lett. 162, 117-122.
(49) Gamboa da Costa, G., Manjanatha, M. G., Marques, M. M., and
Beland, F. A. (2002) Induction of lacI mutations in Big Blue rats
treated with tamoxifen and R-hydroxytamoxifen. Cancer Lett. 176,
37-45.
(50) Chen, T., Gamboa da Costa, G., Marques, M. M., Shelton, S. D.,
Beland, F. A., and Manjanatha, M. G. (2002) Mutations induced
by R-hydroxytamoxifen in the lacI and cII genes of Big Blue
transgenic rats. Carcinogenesis 23, 1751-1757.
(51) 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.
(52) Pathak, D. N., Pongracz, K., and Bodell, W. J . (1996) Activation
of 4-hydroxytamoxifen and the tamoxifen derivative metabolite
E by uterine peroxidase to form DNA adducts: comparison with
DNA adducts formed in the uterus of Sprague-Dawley rats
treated with tamoxifen. Carcinogenesis 17, 1785-1790.
(53) White, I. N. H., Martin, E. A., Mauthe, R. J ., Vogel, J . S.,
Turteltaub, K. W., and Smith, L. L. (1997) Comparisons of the
binding of [14C]radiolabelled tamoxifen or toremifene to rat DNA
using accelerator mass spectrometry. Chem. Biol. Interact. 106,
149-160.
(54) Beland, F. A., McDaniel, L. P., and Marques, M. M. (1999)
Comparison of the DNA adducts formed by tamoxifen and
4-hydroxytamoxifen in vivo. Carcinogenesis 20, 471-477.
(55) Schild, L. J ., Divi, R. L., and Poirier, M. C. (2002) Formation of
tamoxifen-DNA adducts in organs of monkeys dosed orally with
tamoxifen for 30 days. Environ. Mol. Mutagen. 39 (Suppl 33), 56.
(56) Beland, F. A., Churchwell, M. I., Doerge, D. R., Malejka-Giganti,
D., Zhang, X., Divi, R. L., Poirier, M. C., Gamboa da Costa, G.,
and Marques, M. M. (2002) High performance liquid chromatog-
raphy electrospray ionization tandem mass spectrometry analysis
of tamoxifen DNA adducts in rats, monkeys, and humans. Proc.
Am. Assoc. Cancer Res. 43, 343.
(57) 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.
(58) Orton, T. C., and Topham, J . C. (1997) Correspondence regarding
Hemminki, K., et al. Tamoxifen-induced DNA adducts in en-
dometrial samples from breast cancer patients. Cancer Res. 57,
4148.
(59) Shibutani, S., Suzuki, N., Terashima, I., Sugarman, S. M.,
Grollman, A. P., and Pearl, M. L. (1999) Tamoxifen-DNA adducts
detected in the endometrium of women treated with tamoxifen.
Chem. Res. Toxicol. 12, 646-653.
TX020090G