Slow loss of deoxyribose from the N7deoxyguanosine adducts of estradiol-3,4-quinone and hexestrol-3′,4′-quinone.: Implications for mutagenic activity

Article abstract of DOI:10.1016/j.steroids.2004.09.011

A variety of evidence has been obtained that estrogens are weak tumor initiators. A major step in the multi-stage process leading to tumor initiation involves metabolic formation of 4-catechol estrogens from estradiol (E ₂) and/or estrone and further oxidation of the catechol estrogens to the corresponding catechol estrogen quinones. The electrophilic catechol quinones react with DNA mostly at the N-3 of adenine (Ade) and N-7 of guanine (Gua) by 1,4-Michael addition to form depurinating adducts. The N3Ade adducts depurinate instantaneously, whereas the N7Gua adducts depurinate with a half-life of several hours. Only the apurinic sites generated in the DNA by the rapidly depurinating N3Ade adducts appear to produce mutations by error-prone repair. Analogously to the catechol estrogen-3,4-quinones, the synthetic nonsteroidal estrogen hexestrol-3′,4′-quinone (HES-3′, 4′-Q) reacts with DNA at the N-3 of Ade and N-7 of Gua to form depurinating adducts. We report here an additional similarity between the natural estrogen E₂ and the synthetic estrogen HES, namely, the slow loss of deoxyribose from the N7deoxyguanosine (N7dG) adducts formed by reaction of E₂-3,4-Q or HES-3′,4′-Q with dG. The half-life of the loss of deoxyribose from the N7dG adducts to form the corresponding 4-OHE ₂-1-N7Gua and 3′-OH-HES-6′-N7Gua is 6 or 8 h, respectively. The slow cleavage of this glycosyl bond in DNA seems to limit the ability of these adducts to induce mutations.

Full text of DOI:10.1016/j.steroids.2004.09.011

Steroids 70 (2005) 29–35
Slow loss of deoxyribose from the N7deoxyguanosine adducts of
estradiol-3,4-quinone and hexestrol-3ꢀ,4ꢀ-quinone.
Implications for mutagenic activity
Muhammad Saeed, Muhammad Zahid, Sandra J. Gunselman, Eleanor Rogan, Ercole Cavalieri∗
Eppley Institute for Research in Cancer and Allied Diseases, University of Nebraska Medical Center,
986805 Nebraska Medical Center, Omaha, NE 68198-6805, USA
Received 19 April 2004; received in revised form 21 July 2004; accepted 21 September 2004
Available online 23 November 2004
Abstract
A variety of evidence has been obtained that estrogens are weak tumor initiators. A major step in the multi-stage process leading to tumor
initiation involves metabolic formation of 4-catechol estrogens from estradiol (E2) and/or estrone and further oxidation of the catechol estrogens
to the corresponding catechol estrogen quinones. The electrophilic catechol quinones react with DNA mostly at the N-3 of adenine (Ade) and
N-7 of guanine (Gua) by 1,4-Michael addition to form depurinating adducts. The N3Ade adducts depurinate instantaneously, whereas the
N7Gua adducts depurinate with a half-life of several hours. Only the apurinic sites generated in the DNA by the rapidly depurinating N3Ade
adducts appear to produce mutations by error-prone repair. Analogously to the catechol estrogen-3,4-quinones, the synthetic nonsteroidal
estrogen hexestrol-3ꢀ,4ꢀ-quinone (HES-3ꢀ,4ꢀ-Q) reacts with DNA at the N-3 of Ade and N-7 of Gua to form depurinating adducts. We report
here an additional similarity between the natural estrogen E2 and the synthetic estrogen HES, namely, the slow loss of deoxyribose from the
N7deoxyguanosine (N7dG) adducts formed by reaction of E2-3,4-Q or HES-3ꢀ,4ꢀ-Q with dG. The half-life of the loss of deoxyribose from
the N7dG adducts to form the corresponding 4-OHE2-1-N7Gua and 3ꢀ-OH-HES-6ꢀ-N7Gua is 6 or 8 h, respectively. The slow cleavage of this
glycosyl bond in DNA seems to limit the ability of these adducts to induce mutations.
© 2004 Elsevier Inc. All rights reserved.
Keywords: Steroidal and nonsteroidal estrogens; Catechol quinones; 1,4-Michael addition; Depurinating DNA adducts
1. Introduction
the metabolism of estrogens leading to tumor initiation in-
cludes formation of 4-catechol estrogens from estradiol (E2)
and/or estrone. These catechol estrogens are further oxidized
to the corresponding catechol estrogen quinones. The cate-
cholquinonesreactwithDNAattheN-3ofadenine(Ade)and
N-7 of guanine (Gua) to form depurinating adducts [8–11].
These adducts are lost from DNA by destabilization of the
glycosyl bond. The apurinic sites generated in the DNA can
produce mutations by error-prone repair [11,12].
A similar metabolic activation has been found for the
synthetic estrogen hexestrol (HES). The major metabolite
of HES is its catechol [3,13], which can be oxidized to the
catechol quinone. The HES-3ꢀ,4ꢀ-quinone (HES-3ꢀ,4ꢀ-Q) re-
acts with DNA at the N-3 of Ade and N-7 of Gua to form
depurinating adducts analogous to the adducts formed by cat-
The natural and synthetic estrogens have been found to
be carcinogenic in animal model studies [1–5]. The nons-
teroidal synthetic estrogen diethylstilbestrol has also been
shown to be carcinogenic in humans [6]. Estrogens are con-
sidered to be weak tumor initiators [7–9]. One pathway in
Abbreviations: Ade, adenine; dG, 2ꢀ-deoxyguanosine; DMF, N,N-
dimethylformamide; E2, estradiol; E2-3,4-Q, estradiol-3,4-quinone; Gua,
guanine; HES, hexestrol; HES-3ꢀ,4ꢀ-Q, hexestrol-3ꢀ,4ꢀ-quinone; MS/MS,
tandem mass spectrometry; 4-OHE2, 4-hydroxyestradiol; 3ꢀ-OH-HES, 3ꢀ-
hydroxyhexestrol; TFA, trifluoroacetic acid
∗
Corresponding author. Tel.: +1 402 559 7237; fax: +1 402 559 8068.
E-mail address: ecavalie@unmc.edu (E. Cavalieri).
0039-128X/$ – see front matter © 2004 Elsevier Inc. All rights reserved.
doi:10.1016/j.steroids.2004.09.011

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M. Saeed et al. / Steroids 70 (2005) 29–35
Fig. 1. Reaction scheme for the oxidation of 4-OHE2 to E2-3,4-Q and its reaction with dG to form 4-OHE2-1-N7dG, followed by loss of deoxyribose to yield
4-OHE2-1-N7Gua.
Fig. 2. Reaction scheme for the oxidation of 3ꢀ-OH-HES to HES-3ꢀ,4ꢀ-Q and its reaction with dG to form 3ꢀ-OH-HES-6ꢀ-N7dG, followed by loss of deoxyribose
to yield 3ꢀ-OH-HES-6ꢀ-N7Gua.

M. Saeed et al. / Steroids 70 (2005) 29–35
31
Fig. 3. Mass spectrum of the reaction of E2-3,4-Q with dG after 3 h at 22 ◦C. Right inset, MS/MS of 4-OHE2-1-N7dG; left inset, MS/MS of 4-OHE2-1-N7Gua.
echol estrogen-3,4-quinones [14,15]. In this article, we report
the slow loss of deoxyribose from the N7deoxyguanosine
(N7dG) adducts formed by reaction of E2-3,4-Q or HES-
3ꢀ,4ꢀ-Q with dG (Figs. 1 and 2). This is an additional similar-
ity between the natural estrogen E2 and the synthetic estrogen
HES.
separations were monitored at 290 nm for 4-OHE2 and at
277 nm for 3ꢀ-OH-HES.
3.2. Mass spectrometry
To verify the presence and identity of the adducts, reaction
components were analyzed by direct infusion MS and tan-
dem MS (MS/MS), using a MicroMass QuattroMicro triple
quadrupole mass spectrometer (Waters). A 100-␮l aliquot of
the reaction mixture was diluted 1:1 in triple distilled H2O,
then passed through a 0.22 ␮m syringe filter prior to infu-
sion into the mass spectrometer. Flow from the syringe was
5 ␮l/min directed to an interface at 120 ◦C with nitrogen as
both the desolvation and auxiliary gas. Reaction components
were ionized in the positive ion mode with the capillary at
3.5 kV and cone at 25 V. Full-scan spectra between 20 and
700 amu were obtained by combining 100 scans. MS/MS
conditions were optimized for each individual peak identi-
fied in full scan spectra. Argon was used as the collision
gas.
2. Experimental methods
2.1. Chemicals and reagents
Ag2O and DMSO-d6 were purchased from Aldrich Chem-
ical Co. (Milwaukee, WI). 2ꢀ-Deoxyguanosine (dG) was pur-
chased from USB (Cleveland, OH). 4-Hydroxyestradiol (4-
OHE2) was synthesized as previously described [16] and 3ꢀ-
hydroxyhexestrol (3ꢀ-OH-HES) was synthesized by a new
method [17]. Activated MnO2 was prepared as previously de-
scribed [18], by treating concentrated aqueous KMnO4 with
aqueous MnSO4 solution kept at 90 ◦C, until a slight excess
of KMnO4 was present, as indicated by the pink coloration
of the suspension.
3.3. HPLC
Analytical HPLC was conducted on a Waters 2690 Sep-
arations Module equipped with a Waters 996 photodiode
array detector and a reversed phase Phenomenex Luna-2
3. Instrumentation
3.1. UV
˚
C-18 column (250 mm × 4.6 mm, 5 ␮m; 120 A, Torrance,
The UV spectra were obtained during HPLC by using a
Waters 996 photodiode array detector (Milford, MA). HPLC
CA). Preparative HPLC was conducted on a Waters 600E
solvent delivery system equipped with a 996 photodiode

32
M. Saeed et al. / Steroids 70 (2005) 29–35
Fig. 4. Mass spectrum of the reaction of HES-3ꢀ,4ꢀ-Q with dG after 3 h at 22 ◦C. Right inset, MS/MS of 3ꢀ-OH-HES-6ꢀ-N7dG; left inset, MS/MS of 3ꢀ-OH-
HES-6ꢀ-N7Gua.
similar UV spectra. The reactions were run and analyzed in
triplicate.
˚
array detector and YMC ODS-AQ 5-␮m, 120 A column
(20 mm × 250 mm, Morris Plains, NJ).
3.4. Reaction of E2-3,4-Q with dG and loss of
deoxyribose
3.5. Reaction of HES-3ꢀ,4ꢀ-Q with dG and loss of
deoxyribose
To an efficiently stirred suspension of activated MnO2
(61 mg, 0.70 mmol) in 1 ml of dimethylformamide (DMF)
at 0 ◦C was added a solution of 4-OHE2 (20 mg, 0.07 mmol)
in 1 ml of DMF under argon [19]. After stirring for 20 min,
theyellowish-greenestradiol-3,4-quinone(E2-3,4-Q)wasfil-
tered quickly through a syringe filter into a solution of dG
(99 mg, 0.35 mmol) in DMF/H2O (4 ml, 1:3). The final solu-
tion was DMF/H2O (1:1). The pH of the solution was ad-
justed to 4 with acetic acid (200 ␮l), and the progress of
the reaction at room temperature (22 ◦C) was monitored at
different time intervals (0.5, 1, 2, 3, 4, 5, 7, 10, 12, 16,
20, and 24 h). Aliquots at the selected time points were
analyzed quickly by using analytical HPLC monitored at
290 nm. The column was eluted starting with 10% CH3CN
in H2O (0.4% trifluoroacetic acid (TFA) (and then with a
linear gradient to 100% CH3CN in 30 min. The 4-OHE2-1-
N7dG, 4-OHE2-1-N7Gua, and E2-3,4-Q eluted at 10.4, 11.1,
and 17.3 min, respectively, and were analyzed and quanti-
fied at 290 nm by using MillenniumTM software. Quantifi-
cation was accomplished by using standards of E2-3,4-Q
and 4-OHE2-1-N7Gua; the latter served for both 4-OHE2-
1-N7Gua and 4-OHE2-1-N7dG, as both compounds showed
3ꢀ-OH-HES (17 mg, 0.2 mmol) was dissolved in 1.5 ml
of acetone and cooled to 0 ◦C, and 54 mg of Ag2O was
added slowly. The reaction mixture was brought to room
temperature (22 ◦C) and stirred for 30 min. It was mixed
with 1 ml of DMF and the acetone was evaporated. The
yellowish-green quinone was filtered directly into a solu-
tion of dG (83 mg) in DMF/H2O (5 ml, 2:3) at pH 4.0 with
one drop of acetic acid. The final solution was DMF/H2O
(1:1). Formation of the adducts was monitored on the an-
alytical HPLC at 277 nm after 0.5, 1, 2, 3, 4, 5, 7, 9, 11,
17, and 24 h. The column was eluted starting with 10%
CH3CN in H2O (0.4% TFA) for 5 min at a flow rate of
1 ml/min, followed by a linear gradient to 80% CH3CN in
30 min. The 3ꢀ-OH-HES-6ꢀ-N7dG, 3ꢀ-OH-HES-6ꢀ-N7Gua,
and HES-3ꢀ,4ꢀ-Q were eluted at 17, 18, and 26 min, re-
spectively, and were analyzed and quantified at 277 nm by
using MillenniumTM software. Quantification was accom-
plished by using standards of HES-3ꢀ,4ꢀ-Q and 3ꢀ-OH-HES-
6ꢀ-N7Gua; the latter served for both 3ꢀ-OH-HES-6ꢀ-N7Gua
and 3ꢀ-OH-HES-6ꢀ-N7dG, as both compounds showed simi-
lar UV spectra. The reactions were run and analyzed in trip-
licate.

M. Saeed et al. / Steroids 70 (2005) 29–35
33
4. Results
The Michael addition of dG to E2-3,4-Q and to HES-3ꢀ,4ꢀ-
corresponded to the loss of H2O from 4-OHE2 (Fig. 3, right
inset).
Q was conducted at pHs ranging from pH 1 to 7. A pH of 4
was selected for this study because under these conditions the
highest yield of N7Gua adducts was obtained (unpublished
results).
4.2. 3ꢀ-OH-HES-6ꢀ-N7Gua
Reaction of HES-3ꢀ,4ꢀ-Q with dG yielded the labile adduct
3ꢀ-OH-HES-6ꢀ-N7dG, which slowly converted into 3ꢀ-OH-
HES-6ꢀ-N7Gua (Fig. 2). The structure of the latter adduct
was previously demonstrated [14]. After 3 h, an aliquot of the
reaction mixture was directly infused into the mass spectrom-
eter to give the characteristic molecular ions corresponding
to the compounds in the mixture (Fig. 4). The [M + H]+ at
m/z 552 indicated formation of 3ꢀ-OH-HES-6ꢀ-N7dG. Frag-
mentation of this peak by MS/MS yielded the major daugh-
ter ion at m/z 436, corresponding to 3ꢀ-OH-HES-6ꢀ-N7Gua,
which derived from the parent compound minus deoxyri-
bose (Fig. 4, right inset). Other daughter ions were detected
at m/z 268 and 152, which corresponded to dG and Gua,
respectively. These fragments definitively identified the la-
bile adduct 3ꢀ-OH-HES-6ꢀ-N7dG. MS/MS of 3ꢀ-OH-HES-
6ꢀ-N7Gua (Fig. 4, left inset) produced the two fragments at
m/z 300 and 286, which are the fingerprint of this adduct [14].
4.1. 4-OHE2-1-N7Gua
Reaction of E2-3,4-Q with dG yielded the labile adduct
4-OHE2-1-N7dG, which slowly converted into 4-OHE2-1-
N7Gua (Fig. 1). The structure of the latter adduct was pre-
viously demonstrated [19]. After 3 h, an aliquot of the reac-
tion mixture was directly infused into the mass spectrometer
(Fig. 3) to give the [M + H]+ of 4-OHE2-1-N7dG, m/z 554.
The scan of this 3-h mixture also contained a peak at m/z
438, corresponding to 4-OHE2-1-N7Gua, and peaks at m/z
287 and 268, corresponding to E2-3,4-Q and dG, respectively
(Fig. 3). The adduct 4-OHE2-1-N7dG was definitively iden-
tified by MS/MS, in which the fragment at m/z 438 corre-
sponded to 4-OHE2-1-N7Gua and the fragment at m/z 272
Fig. 5. HPLC profile at various time points of the (A) reaction between E2-3,4-Q and dG and (B) reaction between HES-3ꢀ,4ꢀ-Q and dG.

34
M. Saeed et al. / Steroids 70 (2005) 29–35
Fig. 6. Formation and disappearance of 4-OHE2-1-N7dG, formation of 4-OHE2-1-N7Gua and disappearance of E2-3,4-Q over time.
Fig. 7. Formation and disappearance of 3ꢀ-OH-HES-6ꢀ-N7dG, formation of 3ꢀ-OH-HES-6ꢀ-N7Gua and disappearance of HES-3ꢀ,4ꢀ-Q over time.
4.3. Time course of the reaction
increased for 4 h, but then decreased because the adduct was
converted to 3ꢀ-OH-HES-6ꢀ-N7Gua (Fig. 7).
As indicated in Figs. 1 and 5A, the reaction between E2-
3,4-Q and dG initially yielded 4-OHE2-1-N7dG via a 1,4-
Michael addition of the N-7 of dG to the C-1 of E2-3,4-
Q. In the first 5 h, the concentration of E2-3,4-Q decreased
rapidly, concomittantly with the formation of 4-OHE2-1-
N7dG (Fig. 6). This adduct subsequently released the de-
oxyribosemoietyandwasconvertedtothe4-OHE2-1-N7Gua
adduct (Figs. 5A and 6). The concentration of the 4-OHE2-
1-N7dG increased in the first 4 h, but then decreased because
the adduct was converted to 4-OHE2-1-N7Gua (Fig. 6).
Reaction of HES-3ꢀ,4ꢀ-Q with dG proceeded in a similar
manner, with initial formation of 3ꢀ-OH-HES-6ꢀ-N7dG via a
1,4-Michael addition of the N-7 of dG to the C-6ꢀ of HES-
3ꢀ,4ꢀ-Q (Figs. 2, 5B and 7). The 3ꢀ-OH-HES-6ꢀ-N7dG adduct
subsequently lost deoxyribose to yield the 3ꢀ-OH-HES-6ꢀ-
N7Gua adduct. The concentration of 3ꢀ-OH-HES-6ꢀ-N7dG
5. Discussion
The reaction of E2-3,4-Q or HES-3ꢀ,4ꢀ,-Q with dG yielded
the N7dG adduct, which slowly lost the deoxyribose moiety
as a result of destabilization of the glycosyl bond. Formation
and decomposition of the N7dG adducts were demonstrated
by a combination of HPLC analysis of the reactions at var-
ious time points and by MS analysis. The reaction of HES-
3ꢀ,4ꢀ,-Q with dG was approximately 25% less efficient than
the reaction of E2-3,4-Q with dG. The half-life of the N7dG
adduct was approximately 6 h for 4-OHE2-1-N7dG and ap-
proximately 8 h for 3ꢀ-OH-HES-6ꢀ-N7dG. When E2-3,4-Q or
HES-3ꢀ,4ꢀ,-Q is reacted with DNA, the 4-OHE2-1-N3Ade or
3ꢀ-OH-HES-6ꢀ-N3Ade depurinates instantaneously, whereas

M. Saeed et al. / Steroids 70 (2005) 29–35
35
the 4-OHE2-1-N7Gua or 3ꢀ-OH-HES-6ꢀ-N7Gua is released
from the DNA slowly over several hours [15,20].
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The biological effects of the slow release of N7Gua
adducts from DNA in vivo has been demonstrated with E2-
3,4-Q. When mouse skin was topically treated with E2-3,4-Q,
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Despite the formation of N7Gua and N3Ade adducts in sim-
ilar amounts, almost all of the mutations were detected at
Ade sites, suggesting that the rate of depurination is a criti-
cal factor in generating mutations. Therefore, the burden of
mutations by fast depurination has been suggested to be a
critical factor in the induction of cancer-causing mutations
by error-prone misrepair of apurinic sites [12].
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Gross ML, et al. Evidence that a burst of DNA depurination in
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3ꢀ-hydroxyhexestrol. Implications for a unifying mechanism of tu-
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Acknowledgements
This research was supported by U.S. Public Health Ser-
vice grants P01 CA49210 and R01 CA49917 from the Na-
tional Cancer Institute. Core support in the Eppley Institute
is provided by grant P30 CA36727 from the National Cancer
Institute.
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