Molecular characteristics of catechol estrogen quinones in reactions with deoxyribonucleosides

Article abstract of DOI:10.1021/tx960002q

Estrogens can have two roles in the induction of cancer: stimulating proliferation of cells by receptor-mediated processes, and generating electrophilic species that can covalently bind to DNA. The latter role is thought to proceed through catechol estrogen metabolites, which can be oxidized to o-quinones that bind to DNA. Four estrogen-deoxyribonucleoside adducts were synthesized by reaction of estrone 3,4-quinone (E₁-3,4-Q), 17β-estradiol 3,4-quinone (E₂3,4-Q), or estrone 2,3-quinone (E₁-2,3-Q) with deoxyguanosine (dG) or deoxyadenosine (dA) in CH₃CO₂H/H₂O (1:1). Reaction of E₁-3,4-Q or E₂-3,4-Q with dG produced specifically 7-[4- hydroxyestron-1(α,β)-yl]guanine (4-OHE₁-1(α,β)-N7Gua) or 7-[4- hydroxyestradiol-1(α,β)-yl]-guanine (4-OHE₂-1(α,β)-N7Gua), respectively, in 40% yield, with loss of deoxyribose. These two quinones did not react with dA, deoxycytidine, or thymidine. When E₁-2,3-Q was reacted with dG or dA, N²-(2-hydroxyestron-6-yl)deoxyguanosine (2-OHE₁-6-N²dG, 10% yield) and N⁶(2-hydroxyestron-6-yl)deoxyadenosine (2-OHE₁-6-N⁶dA, 80% yield), respectively, were formed. These adducts provide insight into the type of DNA damage that can be caused by o-quinones of the catechol estrogens. The estrogen 3,4-quinones are expected to produce depurinating guanine adducts that are lost from DNA, generating apurinic sites, whereas the 2,3-quinones would form stable adducts that remain in DNA, unless repaired. The adducts reported here will be used as references in studies to elucidate the structure of estrogen adducts in biological systems.

Full text of DOI:10.1021/tx960002q

Chem. Res. Toxicol. 1996, 9, 851-859
851
Articles
Molecu la r Ch a r a cter istics of Ca tech ol Estr ogen
Qu in on es in Rea ction s w ith Deoxyr ibon u cleosid es
Douglas E. Stack,† J aeman Byun,‡ Michael L. Gross,‡ Eleanor G. Rogan,† and
Ercole L. Cavalieri*,†
Eppley Institute for Research in Cancer and Allied Diseases, University of Nebraska Medical
Center, 600 South 42nd Street, Omaha, Nebraska 68198-6805, and Department of Chemistry,
Washington University, One Brookings Drive, St. Louis, Missouri 63130-4899
Received J anuary 2, 1996X
Estrogens can have two roles in the induction of cancer: stimulating proliferation of cells
by receptor-mediated processes, and generating electrophilic species that can covalently bind
to DNA. The latter role is thought to proceed through catechol estrogen metabolites, which
can be oxidized to o-quinones that bind to DNA. Four estrogen-deoxyribonucleoside adducts
were synthesized by reaction of estrone 3,4-quinone (E1-3,4-Q), 17â-estradiol 3,4-quinone (E2-
3,4-Q), or estrone 2,3-quinone (E1-2,3-Q) with deoxyguanosine (dG) or deoxyadenosine (dA) in
CH3CO2H/H2O (1:1). Reaction of E1-3,4-Q or E2-3,4-Q with dG produced specifically 7-[4-
hydroxyestron-1(R,â)-yl]guanine (4-OHE1-1(R,â)-N7Gua) or 7-[4-hydroxyestradiol-1(R,â)-yl]-
guanine (4-OHE2-1(R,â)-N7Gua), respectively, in 40% yield, with loss of deoxyribose. These
two quinones did not react with dA, deoxycytidine, or thymidine. When E1-2,3-Q was reacted
with dG or dA, N2-(2-hydroxyestron-6-yl)deoxyguanosine (2-OHE1-6-N2dG, 10% yield) and N6-
(2-hydroxyestron-6-yl)deoxyadenosine (2-OHE1-6-N6dA, 80% yield), respectively, were formed.
These adducts provide insight into the type of DNA damage that can be caused by o-quinones
of the catechol estrogens. The estrogen 3,4-quinones are expected to produce depurinating
guanine adducts that are lost from DNA, generating apurinic sites, whereas the 2,3-quinones
would form stable adducts that remain in DNA, unless repaired. The adducts reported here
will be used as references in studies to elucidate the structure of estrogen adducts in biological
systems.
monomethoxy derivatives (Scheme 1). These enzymes
are protective, because only nonmethylated CE can be
oxidized to their quinones (CE-Q) by peroxidases and
cytochrome P450 (3, 6-9). It is hypothesized that CE-Q
are the ultimate carcinogenic forms of estrogens, because
these electrophiles can covalently bind to nucleophilic
groups on DNA via a Michael addition. Furthermore,
redox cycling generated by reduction of CE-Q to semi-
quinones and subsequent oxidation back to CE-Q can
generate hydroxyl radicals that can cause additional
DNA damage as proposed by Liehr et al. (8, 10, 11) and
Nutter et al. (12, 13).
In tr od u ction
The role of estrogens in the induction of cancer has
generally been related to stimulation of proliferation by
receptor-mediated processes (1). Estrogens can also play
another important role by generating electrophilic species
that can covalently bind to DNA to initiate cancer (2, 3).
The estrogens 17â-estradiol (E2)1 and estrone (E1), which
are continuously interconverted by 17â-oxidoreductase,
are generally metabolized via two major pathways:
hydroxylation at the 16R-position and at the 2- or
4-position (4, 5). The latter pathway produces catechol
estrogens (CE; Scheme 1).
Several lines of evidence suggest that the 4-hydroxyCE
are critical intermediates in the pathway leading to
estrogen-induced cancer. Malignant renal tumors are
induced in Syrian golden hamsters by treatment with E1
or E2 (14-16), suggesting that E1 and E2 can be procar-
cinogenic compounds. The CE 4-OHE1 and 4-OHE2 also
induce renal tumors in hamsters, whereas the corre-
sponding 2-OH isomers do not (17, 18). Furthermore, an
estrogen 4-hydroxylase activity has been identified not
only in hamster kidney (5, 19, 20) but also in other organs
prone to estrogen-induced cancer, such as rat pituitary
(21), mouse uterus (22), human MCF-7 breast cancer cells
(23), human uterine myometrial tumors (24), and human
breast cancer tissues (25).
In mammalian cells, CE are typically conjugated by
catechol O-methyltransferases (COMT) to give their
* To whom correspondence should be addressed.
† University of Nebraska Medical Center.
‡ Washington University.
X Abstract published in Advance ACS Abstracts, J une 15, 1996.
1
Abbreviations: CE, catechol estrogen(s); CAD, collisionally acti-
vated decomposition; CE-Q, catechol estrogen quinone; COMT, catechol
O-methyltransferase(s); dA, deoxyadenosine; dG, deoxyguanosine;
DMF, dimethylformamide; E1, estrone; E2, 17â-estradiol; E1(E2)-2,3-
Q; estrone (estradiol) 2,3-quinone; FAB MS/MS, fast atom bombard-
ment tandem mass spectrometry; 3-NBA/GLY, 3-nitrobenzyl alcohol/
glycerol; OHE1, hydroxyestrone; OHE2, hydroxyestradiol; 2-OHE1-6-
N6dA, N6-(2-hydroxyestron-6-yl)deoxyadenosine; 2-OHE1-6-N2dG, N2-
(2-hydroxyestron-6-yl)deoxyguanosine; 4-OHE1-1(R,â)-N7Gua, 7-[4-hy-
droxyestron-1(R,â)-yl]guanine; 4-OHE2-1(R,â)-N7Gua, 7-[4-hydroxyes-
tradiol-1(R,â)-yl]guanine; LUMO, lowest unoccupied molecular orbital;
Me2SO, dimethyl sulfoxide; NOE, nuclear Overhauser effect; PDA,
photodiode array; QM, quinone methide.
Adducts formed by direct reaction of CE-Q with DNA
have been compared to those formed after activation of
S0893-228x(96)00002-1 CCC: $12.00 © 1996 American Chemical Society

852 Chem. Res. Toxicol., Vol. 9, No. 5, 1996
Stack et al.
Sch em e 1. Meta bolism of Estr on e a n d 17â-Estr a d iol to o-Qu in on es
reported relative to tetramethylsilane, which was employed as
an internal reference. Nuclear Overhauser effect (NOE) spectra
were obtained by subtracting a reference spectrum, recorded
with the presaturation pulse off-resonance, from a spectrum
recorded with a presaturation pulse on-resonance.
CE by horseradish peroxidase (3). In these studies,
however, the structures of the adducts were not identi-
fied. Reaction of CE-Q with nucleosides would provide
authentic adducts and valuable insight into the mecha-
nism of their formation. In addition, the synthetic
adducts would serve as standard compounds for elucidat-
ing adducts formed by reaction of estrogen metabolites
with DNA.
In this paper the synthesis and characterization of four
estrogen-nucleoside adducts formed by reaction of es-
trone 3,4-quinone (E1-3,4-Q), estradiol 3,4-quinone (E2-
3,4-Q), or estrone 2,3-quinone (E1-2,3-Q) with deoxygua-
nosine (dG) or deoxyadenosine (dA) are reported.
Ca lcu la tion s. All calculations were performed on a Silicon
Graphics Iris Indigo 4000 workstation with the SYBYL 6.2
molecular modeling software (Tripos Assoc., St. Louis, MO).
Structure optimization searches were performed by using the
random conformation search (27) provided by SYBYL. The
number of “hits” for each conformer was set to 6, ensuring a
98.4% chance of finding all possible conformations. Each
conformation was then minimized by using the Tripos force field
with the Powell method of convergence. Energy vs torsion angle
data were obtained by the SYBYL gridsearch feature. Heats
of formation were calculated with the PM3 semiempirical
molecular orbital Hamiltonian (MOPAC 6.0, The Quantum
Chemistry Program Exchange) (28) performed through the
SYBYL interface. Optimizations used the “precise X 100” in
addition to the “MMOK” keyword for molecular mechanics
correction to the guanine amide bond.
F a st Atom Bom ba r d m en t Ta n d em Ma ss Sp ectr om etr y
(F AB MS/MS). Collisionally activated decomposition (CAD)
spectra were obtained by using a VG ZAB-T, a four-sector
tandem mass spectrometer of BEBE design (29). MS1 is a
standard high-resolution double-focusing mass spectrometer
(ZAB) of reverse geometry. MS2 has a prototype Mattauch-
Herzog-type design, incorporating a standard magnet and a
planar electrostatic analyzer of inhomogeneous electric field, a
single-point detector, and array detector. For experiments
reported here, sample quantities were sufficiently large so that
the single-point detector was adequate. Samples were dissolved
in 20 µL of CH3OH, and a 1-µL aliquot was loaded on the probe
along with 1 µL of each matrix: a 1:1 mixture of 3-nitrobenzyl
alcohol and glycerol (3-NBA/GLY), 3-NBA/GLY/LiI, or 3-NBA/
GLY/NaI. A Cs+ ion gun operated at 30 keV was used to desorb
the ions from the probe. The instrument accelerating voltage
was 8 kV.
Exp er im en ta l Section
Ca u tion . Quinones of estrogen catechols are extremely toxic
and were handled according to NIH guidelines (26).
Ch em ica ls. Synthesis of 4-OHE1, 4-OHE2, and 2-OHE1 and
the corresponding quinones was conducted by following the
procedures of Dwivedy et al. (3). The deoxyribonucleosides dG
and dA were purchased from TCI (Portland, OR) as the
monohydrates and dried over P2O5 under vacuum at 110 °C for
48 h prior to use. All other chemicals were purchased from
Aldrich Chemical Co. (Milwaukee, WI) and used without further
purification.
HP LC. HPLC was conducted on a Waters 600E solvent
delivery system equipped with a Waters 990 photodiode array
(PDA) detector interfaced with an APC-IV Powermate computer.
Analytical separations were conducted by using a YMC ODS-
AQ 5-µm, 120-Å column (6.0 × 250 mm) at a flow rate of 1 mL/
min (YMC, Morris Plains, NJ ). The analytical gradient started
with 30% CH3OH in H2O for 5 min, followed by a 60-min linear
gradient (CV6) to 100% CH3OH. Preparative HPLC was
conducted by using a YMC ODS-AQ 5-µm, 120-Å column (20 ×
250 mm) at a flow rate of 6 mL/min. The preparative gradient
started with 40% CH3OH in H2O for 10 min, followed by a linear
gradient (CV6) to 60% CH3OH at 20 min and then held for 15
min. A linear gradient to 100% CH3OH was then conducted
for an additional 10 min, resulting in a total separation time of
45 min.
CAD spectra were obtained after precursor ion activation in
the third field-free region (between MS1 and MS2). Helium was
added to the collision cell (floated at 4 kV) to attenuate the ion
beam by 50%. MS1 was operated at a resolving power of 1000;
MS2 resolving power was set to 1000 (full width at half-height
definition). Ten to fifteen 15-s scans were signal-averaged for
each spectrum. Data acquisition and workup were performed
NMR. NMR spectra were recorded in dimethyl sulfoxide-d6
(Me2SO-d6) at 25 °C on either a Varian XL-300 at 299.938 MHz
or a Varian Unity 500 at 499.835 MHz. Chemical shifts are

Reaction of Estrogen Quinones with Nucleosides
Chem. Res. Toxicol., Vol. 9, No. 5, 1996 853
with a DEC Alpha 3000 workstation equipped with OPUS V
3.1X software and interfaced with the mass spectrometer via a
VG SIOS I unit.
Exact mass measurements were conducted with a Kratos MS-
50 triple analyzer tandem mass spectrometer equipped with a
standard Kratos FAB source (30). The atom beam was 6-7 keV
argon atoms at a total current of 1 mA at the cathode of the
gun. A mixture of CsI and glycerol was used to generate
reference-mass ions for the peak match mode.
6.68 (s, 1H, 1-H), 6.56 (s, 1H, 4-H), 6.36 (t, 1H, J ) 6.7 Hz, 1′-H
[dA]), 5.58 (m, 1H, 6-H), 5.27 (d, 1H, J ) 4.1 Hz, 3′-OH [dA],
exchanged with D2O), 5.17 (t, 1H, J ) 6.0 Hz, 5′-OH [dA],
exchanged with D2O), 4.41 (m, 1H, 3′-H [dA]), 3.88 (m, 1H, 4′-H
[dA]), 3.45-3.59 (m, 2H, 5′-H2 [dA]), 2.73 (q, J ) 6.5 Hz, 1H),
2.38 (dd, J 1 ) 18.5 Hz, J 2 ) 7.5 Hz, 1H), 2.19-2.27 (m, 2H),
1.90-2.16 (m, 4H), 1.84 (t, J ) 9.0 Hz, 1H), 1.73-1.78 (m, 1H),
1.56-1.67 (m, 1H), 1.34-1.49 (m, 3H), 0.81 (s, 3H, 13-CH3). MS,
(M + H)+ C28H34N5O6 calcd 536.25090, found 536.25117.
E1-2,3-Q + d G. A suspension of 2-OHE1 (0.25 mmol) in 5
mL of CH3CN was cooled to -40 °C prior to the addition of
activated MnO2 (2.25 mmol). The suspension was stirred for
10 min and then filtered directly into a stirred solution of dG
(1.30 mmol) in a solvent mixture of 10 mL of CH3CO2H/H2O
(1:1). Aliquots were removed for HPLC analysis at 1, 2, and 5
h to monitor the course of the reaction. After 5 h at room
temperature, solvents were removed under reduced pressure,
and the crude product was dissolved in a 1:1 solvent mixture of
CH3OH/DMF. The product was then isolated via preparative
reverse phase HPLC, as described above, to afford N2-(2-
hydroxyestron-6-yl)deoxyguanosine (2-OHE1-6-N2dG) (10% yield).
2-OHE1-6-N2d G. UV, λmax (nm): 254, 287. 1H NMR (500
MHz): 9.92 (bs, 1H, 1-NH [dG], exchanged with D2O), 8.83 (bs,
2H, 2-OH and 3-OH, exchanged with D2O), 7.91 (s, 1H, 8-H
[dG]), 6.84 (d, 1H, 2-NH [dG], exchanged with D2O), 6.69 (s,
1H, 1-H), 6.64 (s, 1H, 4-H), 6.18 (t, 1H, J ) 6.7 Hz, 1′-H [dG]),
5.29 (d, 1H, J ) 4.0 Hz, 3′-OH [dG], exchanged with D2O), 4.99
(m, 1H, 6-H), 4.87 (t, 1H, J ) 6.0 Hz, 5′-OH [dG], exchanged
with D2O), 4.38 (m, 1H, 3′-H [dG]), 3.81 (m, 1H, 4′-H [dG]), 3.49-
Syn th esis of Estr ogen Nu cleosid e Ad d u cts. E1-3,4-Q or
E2-3,4-Q + d G. A suspension of 4-OHE1 or 4-OHE2 (0.18 mmol)
in 5 mL of CH3CN was cooled to 0 °C prior to the addition of
activated MnO2 (1.18 mmol). The suspension was stirred for
10 min and then filtered directly into a stirred solution of dG
(0.94 mmol) in a solvent mixture of 10 mL of CH3CO2H/H2O
(1:1). Aliquots were removed for HPLC analysis at 1, 2, and 5
h to monitor the course of the reaction. After 5 h at room
temperature, solvents were removed under reduced pressure
and the crude product was dissolved in a 1:1 solvent mixture of
CH3OH/dimethylformamide (DMF). The product was then
isolated via preparative reverse phase HPLC, as described
above, to afford 7-[4-hydroxyestron-1(R,â)-yl]guanine [4-OHE1-
1(R,â)-N7Gua] or 7-[4-hydroxyestradiol-1(R,â)-yl]guanine [4-OHE2-
1(R,â)-N7Gua] (40% yield).
4-OHE1-1(r,â)-N7Gu a . 1H and 13C NMR spectra showed the
product to be a mixture of the designated R and â isomers; the
following spectra are those of the two isomers.
UV, λmax (nm): 211, 291. FTIR (KBr, cm-1): 3430, 2920,
1745, 1700, 1630, 1570, 1480, 1200, 1150, 780. 1H NMR (500
MHz): 10.72 and 10.61 (bs, 1H, 1-NH [Gua], exchanged with
D2O), 9.44 and 9.43 (bs, 1H, 3-OH, exchanged with D2O), 8.50
and 8.49 (bs, 1H, 4-OH, exchanged with D2O), 7.90 and 7.89 (s,
1H, 8-H [Gua]), 6.51 and 6.49 (s, 1H, 2-H), 6.14 and 6.12 (bs,
2H, 2-NH2 [Gua], exchanged with D2O), 2.84 (dd, J 1 ) 17.5 Hz,
J 2 ) 4.0 Hz, 1H), 2.30-2.56 (m, 3H), 1.96-2.02 (m, 1H), 1.83-
1.90 (m, 2H), 1.23-1.61 (m, 4H), 1.10-1.21 (m, 1H), 0.74-0.98
(m, 2H), 0.74 (s, 3H, 13-CH3), 0.45-0.68 (m, 1H). 13C NMR (75
MHz): 162.2, 160.2, 159.9, 153.9, 153.8, 152.9, 152.8, 143.5,
143.2, 143.0, 142.8, 142.1, 142.0, 127.4, 126.3, 126.1, 125.8,
125.7, 113.6, 112.8, 109.3, 108.0, 49.7, 49.4, 47.3, 47.2, 44.2, 43.7,
35.7, 35.1, 32.3, 31.8, 30.7, 25.2, 25.1, 24.4, 24.3, 24.2, 21.1, 13.7,
13.6. MS, (M + H)+ C23H26N5O4 calcd 436.19850, found
436.19863.
3.58 (m, 2H, 5′-H2 [dG]), 2.71 (q, J ) 6.5 Hz, 1H), 2.41 (dd, J 1
)
18.0 Hz, J 2 ) 7.0 Hz, 1H), 2.16-2.28 (m, 2H), 1.98-2.22 (m,
3H), 1.85-1.96 (m, 1H), 1.32-1.18 (m, 6H), 0.85 (s, 3H, 13-CH3).
MS, (M + H)+ C28H34N5O7 calcd 552.24580, found 552.24607.
Resu lts
Syn th esis a n d Str u ctu r e Deter m in a tion of Estr o-
gen Qu in on e Ad d u cts F or m ed w ith Deoxyr ibo-
n u cleosid es. Initial attempts to synthesize nucleoside
adducts of estrogen quinones in aprotic solvents yielded
no adducts. Neither dG nor dA reacted with E1-3,4-Q or
E1-2,3-Q in DMF or Me2SO. In acidic conditions, how-
ever, reaction of estrogen quinones with sulfur nucleo-
philes was previously reported (31). The solvent system
to CH3CO2H/H2O (1:1) facilitated Michael addition of
nucleophilic groups in dG and dA to both E1-3,4-Q and
E1-2,3-Q, to give the products shown in Scheme 2. No
adducts were obtained with deoxycytidine or thymidine.
The quinones were formed in CH3CN at 0 °C for 4-OHE1
and 4-OHE2 and at -40 °C for 2-OHE1 (3) and then
filtered directly into a solution of the nucleoside previ-
ously dissolved in the 1:1 mixture of CH3CO2H/H2O (ca.
pH 2).
Reaction of E1-3,4-Q with dG yielded a product that
eluted as a single peak under several different HPLC
conditions. Reaction yields were typically 40%. The
reaction proceeded to completion after 5 h at room
temperature, and no other adducts were detected. NMR
analysis (detailed discussion below) showed the product
to be a mixture of two conformational isomers attached
to the C1 position of the estrogen A ring and the N7
position of guanine, 4-OHE1-1(R,â)-N7Gua. The genera-
tion of two conformational isomers is the result of a
rotational barrier about the C1-N7Gua bond. Thus, in
one isomer, the guanine moiety is located primarily on
the “R” side of the estrogen ring system, 4-OHE1-1R-
N7Gua, while the other isomer has the guanine moiety
located on the “â” side of the estrogen ring system,
4-OHE1-1â-N7Gua (Figure 1). Rotation from the R
isomer to the â isomer would involve the C8-Gua proton
4-OHE2-1(r,â)-N7Gu a . 1H NMR spectrum showed the prod-
uct to be a mixture of the R and â isomers; the following spectra
are those of the two isomers.
UV, λmax (nm): 211, 292. 1H NMR (500 MHz): 10.72 and
10.64 (bs, 1H, 1-NH [Gua], exchanged with D2O), 9.38 and 9.36
(bs, 1H, 3-OH, exchanged with D2O), 8.44 and 8.43 (bs, 1H,
4-OH, exchanged with D2O), 7.89 and 7.88 (s, 1H, 8-H [Gua]),
6.48 and 6.46 (s, 1H, 2-H), 6.13 and 6.11 (bs, 2H, 2-NH2 [Gua],
exchanged with D2O), 4.39 and 4.35 (d, J ) 3.7 Hz, 1H, 17â-
OH), 4.08 (m, 1H, 17R-H), 2.79 (dd, J 1 ) 17.0 Hz, J 2 ) 4.0 Hz,
1H), 2.30-2.56 (m, 3H), 1.75-1.87 (m, 1H), 1.66-1.75 (m, 1H),
1.46-1.57 (m, 1H), 1.16-1.42 (m, 4H), 0.74 (s, 3H, 13-CH3),
0.48-0.58 (m, 1H). MS, (M + H)+ C23H28N5O4 calcd 438.21410,
found 438.21410.
E1-2,3-Q + d A. A suspension of 2-OHE1 (0.18 mmol) in 5
mL CH3CN was cooled to -40 °C prior to the addition of
activated MnO2 (1.18 mmol). The suspension was stirred for
10 min and then filtered directly into a stirred solution of dA
(0.94 mmol) in a solvent mixture of 10 mL of CH3CO2H/H2O
(1:1). Aliquots were removed for HPLC analysis at 1, 2, and 5
h to monitor the course of the reaction. After 5 h at room
temperature, solvents were removed under reduced pressure,
and the crude product was dissolved in a 1:1 solvent mixture of
CH3OH/DMF. The product was then isolated via preparative
reverse phase HPLC, as described above, to afford N6-(2-
hydroxyestron-6-yl)deoxyadenosine (2-OHE1-6-N6dA) (80% yield).
2-OHE1-6-N6d A. UV, λmax (nm): 207, 276. 1H NMR (300
MHz): 8.61 (s, 1H, OH, exchanged with D2O), 8.57 (s, 1H, OH,
exchanged with D2O), 8.31 and 8.27 (s and bs, 2H, 2-H and 8-H
[dA]), 7.94 (d, 2H, J ) 9.0 Hz, 6-NH [dA], exchanged with D2O),

854 Chem. Res. Toxicol., Vol. 9, No. 5, 1996
Stack et al.
NMR) of 60% â and 40% R (assignment of the R and â
isomers is discussed below). The predominance of the â
isomers indicates formation of this adduct is under
kinetic control, since calculations show the R isomer is
thermodynamically more stable than the â isomer. Mo-
lecular mechanical calculations, ∆ER,â ) -5.9 kcal/mol,
and semiempirical quantum mechanical calculations,
∆Hf ) -2.9 kcal/mol, show the R isomer to be the more
R,â
stable form. Attack of the nitrogen nucleophile at C1 is
the result of a 1,4-addition with respect to the C3
carbonyl. This is in contrast to sulfur nucleophiles, which
attack at the C2 position, a 1,6-addition with respect to
the C4 carbonyl (31). Molecular orbital calculations
(semiempirical, PM3 Hamiltonian) (28) have shown that,
in the neutral species, C1 bears more positive charge than
C2 and that the lowest unoccupied molecular orbital
(LUMO) has a higher coefficient at C2 than C1.2 Thus,
soft electrophiles, such as thiols, will attack at C2 (32),
while harder electrophiles, such as nitrogen and oxygen,
will attack C1.2 When molecular orbital calculations are
done on protonated E1-3,4-Q (i.e., acidic conditions), the
regioselective attack on C1 by hard electrophiles is
enhanced.2
E1-3,4-Q did not react with dA. With this nucleoside,
only gradual decomposition of the quinone was observed.
Reaction of E2-3,4-Q with dG and dA produced the same
results as E1-3,4-Q.
Reaction of E1-2,3-Q with dG yielded the product
2-OHE1-6-N2dG. The product is a result of 1,6-addition
to the quinone after initial tautomerization of E1-2,3-Q
to the quinone methide (QM; Scheme 2). The reaction
yield was low, ca. 10%. Reaction of E1-2,3-Q with dA
produced a similar product, 2-OHE1-6-N6dA, but in much
higher yields of 80%. Recently, investigation into the
isomerization of o-quinones to quinone methides has been
published (33, 34). The o-quinone of 2,3-dihydroxy-
5,6,7,8-tetrahydronaphthalene, in which the A, B rings
are equivalent to 2-OHE1, was shown to be more stable
by 3.8 kcal/mol when tautomerized to the QM (34).
Although the o-quinone is formed initially, it isomerizes
to the more electrophilic QM upon standing in CH3CN
for extended periods of time (34). Calculations on
thermodynamic energies of E1-2,3-Q and E1-2,3-QM6
show they are very close in energy. Molecular mechan-
ical calculations show the QM more stable by 0.3 kcal/
mol, while quantum mechanical calculations (semiem-
pirical, PM3 Hamiltonian) reveal the o-quinone to be
more stable by 1.3 kcal/mol. To study the possibility of
E1-2,3-Q to E1-2,3-QM tautomerization, E1-2,3-Q was
F igu r e 1. Structure of 4-OHE1-1R-N7Gua and 4-OHE1-1â-
N7Gua along with a graph of energy vs torsion angle (C10, C1,
N7Gua, and C8Gua).
Sch em e 2. Rea ction of Estr ogen Qu in on es w ith
d G a n d d A
1
generated in CH3CN-d3 and its H NMR spectrum was
monitored over time. The initial spectrum showed the
presence of only the o-quinone, and the spectrum re-
mained unchanged for 2 days. Even with the addition
of CF3CO2H-d to help catalyze the tautomerization, no
change in the o-quinone spectrum was observed after
standing for 2 weeks. This experiment indicates that in
CH3CN the o-quinone form of E1-2,3-Q is more stable.
However, reaction of E1-2,3-Q with dG and dA resulted
in 6-substituted products, indicating that only the E1-
2,3-QM isomer, present in small quantities, reacts with
these nucleosides.
Because acidic conditions were used to generate these
estrogen-nucleoside adducts, an investigation was con-
ducted of the effects of pH on the stability of both the
occupying the same van der Waals radius as the C11
protons of the estrogen B ring. A graph of energy as a
function of the torsion angle about C10, C1, N7-Gua, and
C8-Gua (Figure 1) shows a rotation barrier of 551 kcal/
mol. The two isomers were formed in a ratio (via 1H
2
Unpublished results (D. Stack and E. Cavalieri).

Reaction of Estrogen Quinones with Nucleosides
Chem. Res. Toxicol., Vol. 9, No. 5, 1996 855
Ta ble 1. Sta bility of E1-3,4-Q a n d E1-2,3-Q a t Va r iou s p H
the C1 proton and the C11 protons have been demon-
strated for the parent compound, 4-OHE1, and other
estrogen adducts substituted at C2 (35). No NOE effect
was seen between the resonance signals at 6.51/6.49 ppm
(2-H) and other protons in the aliphatic region. More-
over, a strong NOE effect was seen between the 3-OH
proton (9.44 and 9.43 ppm) and both resonance signals
at 6.51 and 6.49 ppm. A significant upfield shift of
several aliphatic protons, 1.10-1.21 (m, 1H), 0.74-0.98
(m, 2H), 0.45-0.68 (m, 1H), was also observed (not
shown), whereas the parent compound, 4-OHE1, has no
protons in this region (with the exclusion of the 13-CH3
singlet). These protons are likely to be the ones at C11
or C12 since perpendicular attachment at C1 of the
guanine ring (Figure 1) would produce shielding to these
protons. A similar upfield shift has been seen for adenine
and guanine adducts at C-12 in the benz[a]anthracene
compounds (36). Loss of the deoxyribose moiety is
evidence for nucleophilic attack at N7 of dG. In fact,
substitution at N7 destablilizes the glycosidic bond (36-
38), resulting in the loss of deoxyribose and its signals
in the aliphatic region of the NMR spectrum. The
presence of the 2-NH2 proton resonance at 6.12 ppm, as
well as the C-8 proton resonance at 7.90 ppm (Figure 2A),
indicates that these two nucleophilic sites in the guanine
moiety are not substituted, thereby corroborating sub-
stitution at N7. 13C NMR also confirmed the presence
of two isomers. One isomer would generate 11 carbon
signals in the aromatic region, but 22 were observed. In
the aliphatic region, 12 signals would be seen if one
isomer were present, but 19 were observed, with 5 carbon
atoms of the two isomers having the same chemical shift.
Additional NOE experiments allowed not only the
validation of two conformational isomers, but also as-
signment of the R and â forms. Molecular modeling
(Figure 1) showed that the distance between the proton
on C2 and C8-Gua differs between the R and â isomers,
3.03 and 4.23 Å, respectively. However, the distance
between the protons on 3-OH and C2 are very similar,
3.60 and 3.62 Å, respectively. Therefore, the NOE
between the protons on C2 and C8-Gua, via irradiation
of the C8-Gua protons, should change the ratio of the C2
protons in the NOE difference spectrum. In contrast, the
NOE effect between the protons on 3-OH and C2, via
irradiation of the 3-OH protons, should be equal between
the R and â isomers, and consequentially, the ratio of the
C2 protons should remain unchanged. Three sets of NOE
spectra are shown in Figure 3, with the “normal” 1D
spectrum (presaturation off-resonance) placed above the
difference spectrum displaying the NOE effect. When the
3-OH proton (9.44 and 9.43 ppm) is irradiated, the NOE
effect seen on the C2 protons gives the same ratio
between the resonance signals at 6.51 and 6.49 ppm
(Figure 3A). When the C8-Gua proton (7.91 and 7.90
ppm) is irradiated, the ratio of the two peaks changes
such that the peak at 6.49 is now the major peak (Figure
3B) and hence belongs to the R isomer since the distance
between these protons is smaller than that of the â
isomer. When the C2 proton (6.51 and 6.49 ppm) is
irradiated, the ratio of the 3-OH protons (9.44 and 9.43
ppm) remains unchanged, while the ratio of the C8-Gua
protons (7.91 and 7.90 ppm) is reversed such that the
peak at 7.91 ppm (R isomer) is predominant (Figure 3C).
Thus, the major isomer is the â form.
t1/2 (min)b
pHa
E1-3,4-Qc,d
E1-2,3-Qe
9
7
5
3
<5
110
190
390
<5
110
110
70
a
pH was adjusted with the following 0.1 M buffers: pH 9, Tris-
b
HCl; pH 7, distilled H2O; pH 5, Trizma; pH 3, citric acid. t1/2 is
defined as the time when 1/2 of the original quinone concentration
was observed by HPLC. The original concentration was estab-
lished by injection of an aliquot of the original quinone solution
(0.02 mmol in CH3CN). c Quinones (0.02 mmol) were synthesized
in 2 mL of CH3CN and placed in 10 mL of aqueous buffer.
d
Sampling times (min) are as follows: pH 3: 56, 156, 206, 306,
356, 406; pH 5: 65, 125, 185, 245, 305; pH 7: 5, 40, 80, 130, 190;
pH 9: 5. e Sampling times (min) are as follows: pH 3: 35, 85, 135,
185; pH 5: 3, 53, 158, 208; pH 7: 15, 30, 50, 95, 195; pH 9: 5.
F igu r e 2. NMR spectra of the (A) 4-OHE1-1(R,â)-N7Gua and
(B) 4-OHE2-1(R,â)-N7Gua adducts.
3,4- and 2,3-quinones. E1-3,4-Q showed a marked in-
crease in stability as the pH was lowered from 7 to 3
(Table 1), whereas E1-2,3-Q did not. The 3,4- and 2,3-
quinones decomposed rapidly under mildly basic condi-
tions. Both quinones were relatively stable, with a t1/2
of 110 min at pH 7, indicating a prolonged presence, if
formed in vivo.
(A) 4-OHE1-1(r,â)-N7Gu a . In the NMR spectrum of
4-OHE1-1-N7Gua (Figure 2A), the presence of two sets
of peaks indicates a mixture of two distinct chemical
species. One isomer is formed in greater amount, ap-
proximately 60:40. Initially, a mixture of regioisomers
at the C1 and C2 positions formed by attack of the N7 of
dG was thought to account for the two chemical species.
However, careful analysis by NOE showed that the N7
of guanine is bonded at the C1 position in both isomers,
4-OHE1-1(R,â)-N7Gua (Figure 1). NOE effects between
FAB of 4-OHE1-1(R,â)-N7Gua produces an [M + H]+
ion of m/ z 436 and an [M + Li]+ ion of m/ z 442. The
exact mass is consistent with the proposed elemental

856 Chem. Res. Toxicol., Vol. 9, No. 5, 1996
Stack et al.
F igu r e 3. Determination of R and â isomers of 4-OHE1-1-N7Gua by NOE irradiation. (A) Top spectrum: 1D with presaturation
off-resonance. Bottom spectrum: NOE difference spectrum, irradiation of the 3-OH proton (9.44 and 9.43 ppm). (B) Top spectrum:
1D with presaturation off-resonance. Bottom spectrum: NOE difference spectrum, irradiation of the C8-Gua proton (7.91 and 7.90
ppm). (C) Top spectrum: 1D with presaturation off-resonance. Bottom spectrum: NOE difference spectrum, irradiation of the C2
proton (6.51 and 6.49 ppm).
analogous ion is observed at m/ z 158 in the CAD
spectrum of the [M + Li]+ ion, suggesting that the lithium
is bound to the nucleobase in the precursor ion.
Because the bonding of the nucleobase and steroid is
strong, most fragment ions are generated by charge-
remote cross-ring cleavages (Figure 4), as was previously
established for simpler steroids (39). The m/ z 312 ion
is formed by the loss of C8H12O from charge-remote
cleavages of the C9-C11 and C8-C14 bonds. The other
fragment ions at m/ z 380, 312, and 272 are also produced
by two ring C-C bond cleavages. The ion of m/ z 286 is
F igu r e 4. Portion of the CAD mass spectrum of [M + H]+ ion
formed by two-bond cleavages; it is not, however, the
of m/ z 436 from 4-OHE1-1(R,â)-N7Gua.
radical cation of the steroid, as was expected on the basis
of the CAD spectra of polycyclic aromatic hydrocarbon
composition. CAD spectra of the [M + H]+ and [M + Li]+
adducts (36-38). The formation of the ion of m/ z 298 is
more complex and is initiated by ring opening of the C
ring, followed by hydrogen rearrangement from the
hydroxy group at C-4 of the A ring and loss of C9H14O.
(B) 4-OHE2-1(r,â)-N7Gu a . The NMR spectrum of
4-OHE2-1(R,â)-N7Gua (Figure 2B) is similar to that of
4-OHE1-1(R,â)-N7Gua, with the addition of signals for
the 17R-proton at 4.08 ppm and the 17â-OH proton at
4.38 ppm.
FAB of 4-OHE2-1(R,â)-N7Gua produces an [M + H]+
species of m/ z 438 and an [M + Li]+ ion of m/ z 444. The
exact mass is consistent with the proposed elemental
composition. CAD spectra of the [M + H]+ and [M + Li]+
ions contain a series of ions of the same mass as observed
in the CAD spectra of the [M + H]+ and [M + Li]+ ions,
respectively, of 4-OHE1-1(R,â)-N7Gua, supporting the
hypothesis that the charge is localized on the DNA base
and the fragmentations occur at the B, C, and D rings
ions were investigated to obtain molecular structure
information. Upon collisional activation, the [M + H]+
ions decompose to form major fragment ions of m/ z 418
(M + H - H2O)+, m/ z 380 (C20H22O3N5)+, m/ z 312
(C15H14O3N5)+, m/ z 298 (C14H12O3N5)+, m/ z 286
(C13H12O3N5)+, m/ z 272 (C12H10O3N5)+, m/ z 152 (Gua +
H)+, and m/ z 135 (Gua + H - NH3)+ (Figure 4). The [M
+ Li]+ ions dissociate to produce similar fragment ions
as for the [M + H]+ ions, except those ions containing
the guanine moiety shift by six mass units to higher
mass.
A few fragment ions are formed by simple cleavage at
a protonated site. The ion of m/ z 418 is formed by the
loss of a water molecule presumably upon protonation
of an OH group. The ion of m/ z 152, which is strong
evidence for the presence of guanine, is produced by
protonation at N7 of the guanine, followed by hydride
rearrangement and cleavage of the C-N bond. An

Reaction of Estrogen Quinones with Nucleosides
Chem. Res. Toxicol., Vol. 9, No. 5, 1996 857
F igu r e 6. The CAD mass spectrum of [M + H]+ ion of m/ z
536 from 2-OHE1-6-N6dA.
to the nucleobase. The product ions of m/ z 136 and 117
are protonated adenine and deoxyribose, respectively.
Collisional activation of the ion of m/ z 420, which is the
protonated 2-OHE1-Ade adduct without the ribose, gen-
erates three major fragment ions at m/ z 403 (by loss of
NH3), 285 (2-OHE1 carbocation), and 136 (protonated
adenine). No ion for the deoxyribose (m/ z 117) moiety
was observed.
The CAD spectrum of the [M + Na]+ ion was examined
to determine the location of the charge site of this adduct
when the sample desorbs upon FAB. The major CAD is
the formation of [Ade + Na]+ at m/ z 157. As observed
for OHE1 and OHE2 adducts, other fragment ions that
contain the nucleobase shift by 22 mass units, suggesting
that the sodium ion (like the lithium ion in the previous
examples) is bound to the adenine.
F igu r e 5. NMR spectra of the (A) 2-OHE1-6-N2dG and (B)
2-OHE1-6-N6dA adducts.
(D) 2-OHE1-6-N2d G. The NMR spectrum of 2-OHE1-
6-N2dG (Figure 5B) is similar to that of the 2-OHE1-6-
N6dA with signals corresponding to the deoxyribose
moiety, the C-1 and C-4 protons of the A ring, and the
2-NH of dG coupled to the C-6 position of the B ring.
Again, attack at the prochiral C-6 results in formation
of two diastereoisomers.
remote from the charge site. Exceptions may be ions
formed by losses of water.
(C) 2-OHE1-6-N6d A. Two key structural features are
reflected in the NMR spectrum of 2-OHE1-6-N6dA (Figure
5A). First, the presence of the aliphatic signals corre-
sponding to the deoxyribose moiety suggests substitution
at the 6-NH2 position of dA, because substitution at the
other two nucleophilic groups of dA, N-3 and N-7, leads
to loss of the deoxyribose moiety by depurination (36-
38). This is confirmed by the lack of the signals at 7.33
corresponding to the 6-NH2 in dA (not shown). Instead,
a doublet is found at 7.94 ppm (exchangeable with D2O)
that corresponds to the 6-NH group attached to the C-6
position of the 2-OHE1 moiety. Evidence for substitution
at C-6 is derived from the two-dimensional chemical shift
correlation spectroscopy technique, which shows the C-6
proton resonance at 5.58 ppm coupled with the 6-NH (dA)
proton at 7.94 ppm. Signals for both the 1- and 4-protons
on the A ring are still present, further supporting the
proposed structure. The protons at C-6 are prochiral, and
attack of the dA nucleophile leads to a mixture of
diastereoisomers in a ratio of 4:1. However, assignment
of the two diastereoisomers has yet to be established.
FAB of 2-OHE1-6-N6dA generates an [M + H]+ species
of m/ z 536 and an [M + Na]+ ion of m/ z 558. Upon
collisonal activation, the [M + H]+ ion yields diagnostic
fragment ions of m/ z 420, 285, 252, 136, and 117 (Figure
6). The fragment ion of m/ z 420 is produced by hydrogen
transfer from the deoxyribose to the adenine moiety,
followed by elimination of C5H8O3. This ion is formally
the [M + H]+ species of the modified base. The formation
of the ion of m/ z 285, which is the 2-OHE1 cation, results
from the cleavage between the benzylic C-6 of the 2-OHE1
moiety and the N6 of the exocyclic amino group of dA,
whereas the ion of m/ z 252, protonated dA, is produced
by the same bond cleavage with hydrogen rearrangement
FAB of 2-OHE1-6-N2dG produces two major ions of m/ z
552 [M + H]+ and of m/ z 436 (protonated 2-OHE1-6-N2-
Gua). The latter ion arises by hydrogen transfer from
the sugar moiety to the base, followed by elimination of
C5H8O3. The ion of m/ z 574 [M + Na]+ is also present
in the spectrum. Collisional activation of the ion of m/ z
574 produces three major fragment ions at m/ z 458
[modified Gua + Na]+, 290 [Gua + deoxyribose + Na]+,
and 174 [Gua + Na]+. The metal ion binds to the
nucleobase as in the dA adduct. The CAD spectrum of
the [M + H]+ ion contains diagnostic fragment ions of
m/ z 436 (modified Gua), 285 (2-OHE1 cation), 268
(protonated dG), 152 [Gua + H]+, and 117 (deoxyribose
moiety). Most fragment ions are produced by simple
cleavage, often accompanied by hydrogen rearrangement.
The ion of m/ z 436 is collisionally dissociated to give two
distinctive fragments: protonated Gua and the 2-OHE1
carbocation.
Discu ssion
The catechol pathway in the metabolism of estrogens
can lead to ultimate carcinogenic species, namely, the
catechol estrogen quinones. The reaction of E1-2,3-Q, E1-
3,4-Q, and E2-3,4-Q with deoxyribonucleosides has been
investigated with the purpose of providing insight into
the possible modes of binding of these intermediates to
DNA. At the same time, authentic adducts have been
prepared for use as standards in the elucidation of the

858 Chem. Res. Toxicol., Vol. 9, No. 5, 1996
Stack et al.
(2) Liehr, J . G. (1990) Genotoxic effects of estrogens. Mutat. Res.
238, 269-276.
(3) Dwivedy, I., Devanesan, P., Cremonesi, P., Rogan, E., and
Cavalieri, E. (1992) Synthesis and characterization of estrogen
2,3- and 3,4-quinones. Comparison of DNA adducts formed by
the quinones versus horseradish peroxidase-activated catechol
estrogens. Chem. Res. Toxicol. 5, 828-833.
(4) Ball, P., and Knuppen, R. (1980) Catechol oestrogens (2- and
4-hydroxyestrogens): Chemistry, biogenesis, metabolism, occur-
rence and physiological significance. Acta Endocrinol. (Copen-
hagen) 93 (Suppl. 232), 1-127.
(5) Zhu, B. T., Bui, Q. D., Weisz, J ., and Liehr, J . G. (1994) Conversion
of estrone to 2- and 4-hydroxyestrone by hamster kidney and liver
microsomes: Implications for the mechanism of estrogen-induced
carcinogenesis. Endocrinology 135, 1772-1779.
(6) Kalyanaraman, B., Sealy, R. C., and Sivarajah, K. (1984) An
electron spin resonance study of o-semiquinones formed during
the enzymatic and autoxidation of catechol estrogens. J . Biol.
Chem. 259, 14018-14022.
(7) Kalyanaraman, B., Felix, C. C., and Sealy, R. C. (1985) Semi-
quinone anion radicals of catechol (amine)s, catechol estrogens,
and their metal ion complexes. Environ. Health Perspect. 64,
185-198.
(8) Liehr, J . G., Ulubelen, A. A., and Strobel, H. W. (1986) Cyto-
chrome P-450-mediated redox cycling of estrogens. J . Biol. Chem.
261, 16865-16870.
(9) Roy, D., Bernhardt, A., Strobel, H. W., and Liehr, J . G. (1992)
Catalysis of the oxidation of steroid and stilbene estrogens to
estrogen quinone metabolites by the beta-naphthoflavone-induc-
ible cytochrome P450 IA family. Arch. Biochem. Biophys. 296,
450-456.
(10) Liehr, J . G., and Roy, D. (1990) Free radical generation by redox
cycling of estrogens. Free Radical Biol. Med. 8, 415-423.
(11) Liehr, J . G. (1994) Mechanisms of metabolic activation and
inactivation of catecholestrogens: A basis of genotoxicity. Poly-
cyclic Aromat. Compd. 6, 229-239.
(12) Nutter, L. M., Ngo, E. O., and Abul-Hajj, Y. J . (1991) Character-
ization of DNA damage induced by 3,4-estrone-o-quinone in
human cells. J . Biol. Chem. 266, 16380-16386.
(13) Nutter, L. M., Wu, Y.-Y., Ngo, E. O., Sierra, E. E., Gutierrez, P.
L., and Abul-Hajj, Y. J . (1994) An o-quinone form of estrogen
produces free radicals in human breast cancer cells: Correlation
with DNA damage. Chem. Res. Toxicol. 7, 23-28.
(14) Li, J . J ., Li, S. A., Klicka, J . K., Parsons, J . A., and Lam, L. K. T.
(1983) Relative carcinogenic activity of various synthetic and
natural estrogens in the Syrian hamster kidney. Cancer Res. 43,
5200-5204.
(15) Kirkman, H. (1959) Estrogen-induced tumors of the kidney in the
Syrian hamster. Natl. Cancer Inst. Monogr. No. 1, 1-59.
(16) Liehr, J . G. (1983) 2-Fluoroestradiol: Separation of estrogenicity
from carcinogenicity. Mol. Pharmacol. 23, 278-281.
(17) Liehr, J . G., Fang, W. F., Sirbasku, D. A., and Ari-Ulubelen, A.
(1986) Carcinogenicity of catechol estrogens in Syrian hamsters.
J . Steroid. Biochem. 24, 353-356.
(18) Li, J . J ., and Li, S. A. (1987) Estrogen carcinogenesis in Syrian
hamster tissues: Role of metabolism. Fed. Proc. 46, 1858-1863.
(19) Weisz, J ., Bui, Q. D., Roy, D., and Liehr, J . G. (1992) Elevated
structure of biologically-formed DNA adducts.
The 2,3- and 3,4-quinones display different chemistry
toward the nucleophiles dG and dA. The most striking
feature, with implication for DNA damage, is that the
3,4-quinones form N7Gua adducts in which the glycosidic
bond of dG is destabilized, leading to formation of the
E1- or E2-1(R,â)-N7Gua adducts. In contrast, the 2,3-qui-
nones bind at the exocyclic amino group of dG or dA,
allowing the deoxyribose moiety to remain attached to
the adduct.
These results have several implications for DNA dam-
age. First, the o-quinones of estrogens are capable of
binding directly to the nucleophilic groups of DNA bases;
second, the 3,4-quinones of estrogens produce depurinat-
ing adducts that are lost from DNA, generating apurinic
sites in the DNA; and third, the 2,3-quinones form stable
adducts that remain bound to DNA if not repaired. In
fact, reaction of 2,3- and 3,4-quinones with DNA or CE
activated by horseradish peroxidase in the presence of
DNA affords differential formation of stable adducts, as
evidenced by 32P-postlabeling analysis (3). The 2,3-
quinones form 10-50 times higher levels of stable
adducts than the 3,4-quinones. The 3,4-quinones and the
enzymically-activated 4-OHE1 and 4-OHE2 form almost
exclusively the depurinating E1(E2)-1(R,â)-N7Gua ad-
ducts both in vitro and in the rat mammary gland.3
In the hamster kidney tumor model, 4-OHE1 and
4-OHE2 are carcinogenic, whereas the corresponding
2-OH isomers are not (17, 18). The elevated expression
of estrogen 4-hydroxylase activity in human and animal
tissues susceptible to estrogen-induced tumors (5, 19-
25) is a further indication that the 4-catechol pathway
may be involved in tumor initiation by estrogens. The
carcinogenicity of the 4-OH estrogens may be explained
by the formation of depurinating adducts with the 3,4-
quinones. This activity contrasts with the noncarcino-
genicity of the 2-OH estrogens, which form only stable
adducts. A similar relationship has been established
between formation of depurinating DNA adducts by
polycyclic aromatic hydrocarbons and induction of onco-
genic mutations in mouse skin papillomas induced by
these compounds (40).
In conclusion, the adducts described here provide
insight into the type of DNA damage possible when CE-Q
are generated in vitro and in vivo and will be used in
studies designed to elucidate the structure of estrogen
adducts in biological systems.
4-hydroxylation of estradiol by hamster kidney microsomes:
A
potential pathway of metabolic activation of estrogens. Endocri-
nology 131, 655-661.
(20) Ashburn, S. P., Han, X., and Liehr, J . G. (1993) Microsomal
hydroxylation of 2- and 4-fluoroestradiol to catechol metabolites
and their conversion to methyl esters: Catechol estrogens as
possible mediators of hormonal carcinogenesis. Mol. Pharmacol.
43, 534-541.
(21) Bui, Q. D., and Weisz, J . (1988) Identification of microsomal,
organic hydroperoxide-dependent catechol estrogen formation:
Comparison with NADPH dependent mechanisms. Pharmacology
36, 356-364.
(22) Bunyagidj, C., and McLachlan, J . A. (1988) Catecholestrogen
formation in mouse uterus. J . Steroid Biochem. 31, 795-801.
(23) Spink, D. C., Eugster, H. P., Lincoln, D. W., II, Schuetz, J . D.,
Schuetz, E. G., J ohnson, J . A., Kaminsky, L. S., and Gierthy, J .
F. (1992) 17 Beta-estradiol hydroxylation catalyzed by human
cytochrome P450 1A1: A comparison of the activities induced by
2,3,7,8-tetrachlorodibenzo-p-dioxin in MCF-7 cells with those from
heterologous expression of the cDNA. Arch. Biochem. Biophys.
293, 342-348.
(24) Liehr, J . G., Ricci, M. J ., J efcoate, C. R., Hannigan, E. V.,
Hokanson, J . A., and Zhu, B. T. (1995) 4-Hydroxylation of
estradiol by human uterine myometrium and myoma mi-
crosomes: Implications for the mechanism of uterine tumorigen-
esis. Proc. Natl. Acad. Sci. U.S.A. 92, 9220-9224.
Ack n ow led gm en t. We thank Drs. S.-T. J an and P.
Mulder for synthesis of catechol estrogens. Molecular
modeling studies were conducted in the Molecular Mod-
eling Core Facility, UNMC/Eppley Cancer Center. We
gratefully acknowledge support from USPHS Grant P01
CA49210 and NRSA 1F32 CA65084. Core support for
the Eppley Institute was provided by Grant CA36727
from the National Cancer Institute. The Washington
University Mass Spectrometry Resource is supported by
the NIH National Center for Research Resources (Grant
2P41RR00954).
Refer en ces
(1) Nandi, S., Guzman, R. C., and Yang, J . (1995) Hormones and
mammary carcinogenesis in mice, rats, and humans: A unifying
hypothesis. Proc. Natl. Acad. Sci. U.S.A. 92, 3650-3657.
3
Unpublished results (E. Cavalieri et al.).

Reaction of Estrogen Quinones with Nucleosides
Chem. Res. Toxicol., Vol. 9, No. 5, 1996 859
(25) Liehr, J . G., and Ricci, M. J . (1996) 4-Hydroxylation of estrogens
as marker of human mammary tumors. Proc. Natl. Acad. Sci.
U.S.A. 93, 3294-3296.
(26) NIH Guidelines for the Laboratory Use of Chemical Carcinogens
(1981) NIH Publication No. 81-2385, U.S. Government Printing
Office, Washington, DC.
o-quinones to p-quinone methides: Potential bioactivation mech-
anism for catechols. Chem. Res. Toxicol. 8, 537-544.
(35) Abul-Hajj, Y. J ., Tabakovic, K., and Tabakovic, I. (1995) An
estrogen-nucleic acid adduct. Electroreductive intermolecular
coupling of 3,4-estrone-o-quinone and adenine. J . Am. Chem. Soc.
117, 6144-6145.
(36) RamaKrishna, N. V. S., Cavalieri, E. L., Rogan, E. G., Dolni-
kowski, G. G., Cerny, R. L., Gross, M. L., J eong, H., J ankowiak,
R., and Small, G. J . (1992) Synthesis and structure determination
of the adducts of the potent carcinogen 7,12-dimethylbenz[a]-
anthracene and deoxyribonucleosides formed by electrochemical
oxidation: Models for metabolic activation by one-electron oxida-
tion. J . Am. Chem. Soc. 114, 1863-1874.
(37) Rogan, E., Cavalieri, E., Tibbels, S., Cremonesi, P., Warner, C.,
Nagel, D., Tomer, K., Cerny, R., and Gross, M. (1988) Synthesis
and identification of benzo[a]pyrene-guanine nucleoside adducts
formed by electrochemical oxidation and horseradish peroxidase-
catalyzed reaction of benzo[a]pyrene with DNA. J . Am. Chem.
Soc. 110, 4023-4029.
(38) RamaKrishna, N. V. S., Gao, F., Padmavathi, N. S., Cavalieri, E.
L., Rogan, E. G., Cerny, R. L., and Gross, M. L. (1992) Model
adducts of benzo[a]pyrene and nucleosides formed from its radical
cation and diol epoxide. Chem. Res. Toxicol. 5, 293-302.
(39) Tomer, K. B., and Gross, M. L. (1988) Fast atom bombardment
and tandem mass spectrometry for structure determination:
Remote site fragmentation of steroid conjugates and bile salts.
Biomed. Environ. Mass Spectrom. 15, 89-98.
(40) Chakravarti, D., Pelling, J . C., Cavalieri, E. L., and Rogan, E. G.
(1995) Relating aromatic hydrocarbon-induced DNA adducts and
c-Harvey-ras mutations in mouse skin papillomas: The role of
apurinic sites. Proc. Natl. Acad. Sci. U.S.A. 92, 10422-10426.
(27) Sanders, M., Hounk, K. N., Yun-Dong, W., Still, W. C., Lipton,
M., Chang, G., and Guida, W. C. (1990) Conformations of
cycloheptadecane. A comparison of methods for conformational
searching. J . Am. Chem. Soc. 112, 1419-1427.
(28) Stewart, J . J . P. (1990) MOPAC: A semiempirical molecular
orbitial program. J . Comput.-Aided Mol. Des. 4, 1-105.
(29) Gross, M. L. (1990) Tandem mass spectrometry: Multisector
magnetic instruments. In Methods in Enzymology, Vol. 193, Mass
Spectrometry (McCloskey, J . A., Ed.) pp 131-153, Academic Press,
San Diego.
(30) Gross, M. L., Chess, E. K., Lyon, P. A., Crow, F. W., Evans, S.,
and Tudge, H. (1982) Triple analyzer mass spectrometry for high
resolution MS/MS studies. Int. J . Mass Spectrom. Ion Phys. 42,
243-254.
(31) Abul-Hajj, Y. J ., and Cisek, P. L. (1988) Catechol estrogen adducts.
J . Steroid Biochem. 31, 107-110.
(32) Abul-Hajj, Y. J ., Tabakovic, K., Gleason, W. B., and Ojala, W. H.
(1996) Reactions of 3,4-estrone quinone with mimics of amino acid
side chains. Chem. Res. Toxicol. 9, 434-438.
(33) Bolton, J . L., Acay, N. M., and Vukomanovic, V. (1994) Evidence
that 4-allyl-o-quinones spontaneously rearrange to their more
electrophilic quinone methides: Potential bioactivation mecha-
nism for the hepatocarcinogen safrole. Chem. Res. Toxicol. 7,
443-450.
(34) Iverson, S. L., Hu, Q. L., Vukomanovic, V., and Bolton, J . L. (1995)
The influence of p-alkyl substituent on the isomerization of
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