The equine estrogen metabolite 4-hydroxyequilenin causes DNA single- strand breaks and oxidation of DNA bases in vitro

Article abstract of DOI:10.1021/tx980083l

Premarin (Wyeth-Ayerst) is the estrogen replacement treatment of choice and continues to be one of the most widely dispensed prescriptions in North America. In addition to endogenous estrogens, Premarin contains unsaturated equine estrogens, including equilenin [1,3,5(10),6,8-estrapentaen-3-ol-17- one]. In previous work, we showed that the equilenin metabolite 4- hydroxyequilenin (4-OHEN) can be autoxidized to 4-OHEN-o-quinone which readily entered into a redox couple with the semiquinone radical catalyzed by NAD(P)H, P450 reductase, or quinone reductase, resulting in generation of reactive oxygen species [Shen, L., Pisha, E., Huang, Z., Pezzuto, J. M., Krol, E., Alam, Z., van Breemen, R. B., and Bolton, J. L. (1997) Carcinogenesis 18, 1093-1101]. As oxidative damage to DNA by reactive oxygen species generated by redox active compounds has been proposed to lead to tumor formation, we investigated whether 4-OHEN could cause DNA damage. We treated λ phage DNA with 4-OHEN and found that extensive single-strand breaks could be obtained with increasing concentrations of 4-OHEN as well as increasing incubation times. If scavengers of reactive oxygen species are included in the incubations, DNA could be completely protected from 4-OHEN- mediated damage. In contrast, NADH and CuCl₂ enhanced the ability of 4-OHEN to cause DNA single-strand breaks presumably due to redox cycling between 4- OHEN and the semiquinone radical generating hydrogen peroxide and ultimately copper peroxide complexes. We also confirmed that 4-OHEN could oxidize DNA bases since hydrolysis of 4-OHEN-treated calf thymus DNA and HPLC separation with electrospray MS detection revealed oxidized deoxynucleosides, including 8-oxodeoxyguanosine and 8-oxodeoxyadenosine. Our data suggest that DNA single-strand breaks and oxidation of DNA bases by 4-OHEN could contribute to the carcinogenic mechanism(s) of equine estrogens.

Full text of DOI:10.1021/tx980083l

Chem. Res. Toxicol. 1998, 11, 1105-1111
1105
Th e Equ in e Estr ogen Meta bolite 4-Hyd r oxyequ ilen in
Ca u ses DNA Sin gle-Str a n d Br ea k s a n d Oxid a tion of DNA
Ba ses in Vitr o
Yumei Chen,† Li Shen,† Fagen Zhang,† Serrine S. Lau,‡
Richard B. van Breemen,† Dejan Nikolic,† and J udy L. Bolton*,†
Department of Medicinal Chemistry and Pharmacognosy (M/ C 781), College of Pharmacy, The
University of Illinois at Chicago, 833 South Wood Street, Chicago, Illinois 60612-7231, and Division
of Pharmacology and Toxicology, College of Pharmacy, University of Texas at Austin,
Austin, Texas 78712
Received April 23, 1998
Premarin (Wyeth-Ayerst) is the estrogen replacement treatment of choice and continues to
be one of the most widely dispensed prescriptions in North America. In addition to endogenous
estrogens, Premarin contains unsaturated equine estrogens, including equilenin [1,3,5(10),6,8-
estrapentaen-3-ol-17-one]. In previous work, we showed that the equilenin metabolite
4-hydroxyequilenin (4-OHEN) can be autoxidized to 4-OHEN-o-quinone which readily entered
into a redox couple with the semiquinone radical catalyzed by NAD(P)H, P450 reductase, or
quinone reductase, resulting in generation of reactive oxygen species [Shen, L., Pisha, E.,
Huang, Z., Pezzuto, J . M., Krol, E., Alam, Z., van Breemen, R. B., and Bolton, J . L. (1997)
Carcinogenesis 18, 1093-1101]. As oxidative damage to DNA by reactive oxygen species
generated by redox active compounds has been proposed to lead to tumor formation, we
investigated whether 4-OHEN could cause DNA damage. We treated λ phage DNA with
4-OHEN and found that extensive single-strand breaks could be obtained with increasing
concentrations of 4-OHEN as well as increasing incubation times. If scavengers of reactive
oxygen species are included in the incubations, DNA could be completely protected from
4-OHEN-mediated damage. In contrast, NADH and CuCl2 enhanced the ability of 4-OHEN
to cause DNA single-strand breaks presumably due to redox cycling between 4-OHEN and
the semiquinone radical generating hydrogen peroxide and ultimately copper peroxide
complexes. We also confirmed that 4-OHEN could oxidize DNA bases since hydrolysis of
4-OHEN-treated calf thymus DNA and HPLC separation with electrospray MS detection
revealed oxidized deoxynucleosides, including 8-oxodeoxyguanosine and 8-oxodeoxyadenosine.
Our data suggest that DNA single-strand breaks and oxidation of DNA bases by 4-OHEN
could contribute to the carcinogenic mechanism(s) of equine estrogens.
corresponding deoxynucleoside (8-oxo-dG) using electro-
chemical detection (5). However, several other oxidized
bases could be formed (Figure 1), including 8-oxoadenine
(8-oxo-A), 2-oxoadenine (2-oxo-A), thymine glycol, 5-hy-
droxymethyluracil, and 5-formyluracil (4). Subsequent
replication of the oxidatively damaged DNA can result
in double mutations (6) and single-base substitutions (7).
The excessive production of reactive oxygen species in
breast cancer tissue has been linked to metastasis of
tumors in women with breast cancer (8). The source of
reactive oxygen species has been suggested to be the
result of redox cycling of the endogenous estrogen me-
tabolites, the catechol estrogens (9, 10). Peroxidase/P450-
catalyzed oxidation of these catechols gives o-quinones,
In tr od u ction
Oxidative damage to DNA is thought to play a signifi-
cant role in spontaneous mutagenesis, cancer, aging, and
other human pathologies (1, 2). Redox active xenobiotics
as well as endogenous compounds can spontaneously or
enzymatically generate reactive oxygen species such as
superoxide. Dismutation of superoxide gives hydrogen
peroxide, which through metal-catalyzed Fenton chem-
istry can form the ultimate oxidant, the hydroxyl radical
that could be responsible for DNA oxidation. Alterna-
tively, transition metals such as iron and copper can react
with hydrogen peroxide, generating metal peroxide com-
plexes which can cause DNA strand breaks and oxidation
of DNA bases (3, 4). The most thoroughly studied
oxidized base is 8-oxoguanine (8-oxo-G)1 since a highly
sensitive HPLC method has been developed to detect the
1 Abbreviations: dN, 2′-deoxynucleoside; dG, 2′-deoxyguanosine; dA,
2′-deoxyadenosine; dC, 2′-deoxycytosine; 8-oxo-dG, 8-oxodeoxygua-
nosine; 8-oxo-dA, 8-oxodeoxyadenosine; 2-oxo-dA, 2-oxodeoxyadenosine;
8-oxo-G, 8-oxoguanine; 8-oxo-A, 8-oxoadenine; 2-oxo-A, 2-oxoadenine;
2-OHE, 2-hydroxyestrone, 2,3-dihydroxy-1,3,5(10)-oestratrien-17-one;
4-OHE, 4-hydroxyestrone, 3,4-dihydroxy-1,3,5(10)-oestratrien-17-one;
4-OHEN, 4-hydroxyequilenin, 3,4-dihydroxy-1,3,5(10),6,8-estrapen-
taen-17-one; estrone, 3-hydroxy-1,2,5(10)-oestratrien-17-one; equilenin,
1,3,5(10),6,8-estrapentaen-3-ol-17-one; equilin, 1,3,5(10),7-estratetraen-
3-ol-17-one; ERT, estrogen replacement therapy; P450, cytochrome
P450.
* Address editorial correspondence to Dr. J udy L. Bolton, Depart-
ment of Medicinal Chemistry and Pharmacognosy (M/C 781), College
of Pharmacy, The University of Illinois at Chicago, 833 S. Wood St.,
Chicago, IL 60612-7231. Fax: (312) 996-7107. E-mail: J udy.Bolton@
UIC.edu.
† The University of Illinois at Chicago.
‡ University of Texas at Austin.
S0893-228x(98)00083-6 CCC: $15.00 © 1998 American Chemical Society
Published on Web 08/19/1998

1106 Chem. Res. Toxicol., Vol. 11, No. 9, 1998
Chen et al.
OHEN, Figure 2), and examined how aromatization of
the B ring affects the formation and reactivity of the
equilenin quinoids (25). Unlike the endogenous catechol
estrogens, 4-OHEN rapidly autoxidized to 4-OHEN-o-
quinone which readily entered into a redox couple with
the semiquinone radical catalyzed by NAD(P)H, P450
reductase, or quinone reductase (Figure 2; 25). Signifi-
cant oxygen consumption was also detected, consistent
with in vitro models that have shown that 4-OHEN-o-
quinone increases the amount of oxidative damage to
DNA by 50% compared to control levels (26). Finally,
we showed that the 4-OHEN-semiquinone radical forms
very unusual cyclic adducts with deoxynucleosides and
DNA which may represent one mechanism for equilenin
carcinogenesis (27, 28). We report here the relative
ability of 4-OHEN to cause DNA single-strand breaks as
well as the oxidized bases resulting from reaction of
4-OHEN with DNA under redox cycling conditions.
F igu r e 1. Potential DNA oxidation products.
which have previously been implicated as the ultimate
carcinogens. Redox cycling between the o-quinones and
their semiquinone radicals generates superoxide, hydro-
gen peroxide, and ultimately reactive hydroxyl radicals,
which cause oxidative cleavage of the phosphate-sugar
backbone as well as oxidation of the purine/pyrimidine
residues of DNA (11). In support of this mechanism,
various free radical toxicities have been reported in
hamsters treated with 17â-estradiol, including DNA
single-strand breaks (11, 12), lipid peroxidation (13),
8-oxo-dG formation (10), and chromosomal abnormalities
(14, 15). Moreover, in hamsters treated with 17â-
estradiol, antioxidants such as BHA (16) or ascorbate (17)
significantly reduced the incidence of tumors compared
to estrogen treatment alone. Finally, elevated levels of
oxidized dG and dA have been detected in the breast
tissue of cancer patients compared to controls, consistent
with a major role of reactive oxygen species in cancer
initiation and/or progression (18, 19). Increased levels
of 8-oxo-dG in particular have been associated with a
predictive significance for breast cancer risk assessment
(20).
The equine estrogen equilenin [1,3,5(10),6,8-estrapen-
taen-3-ol-17-one] or its 17â-hydroxylated analogue makes
up 5% of the most widely prescribed estrogen replace-
ment formulation, Premarin (Wyeth-Ayerst), yet there
is very little information on the metabolism of these
estrogens either in animal models (21-24) or in women.
It is known that treating hamsters for 9 months with
either estrone, equilin and equilenin, or sulfatase-treated
Premarin resulted in 100% tumor incidences and abun-
dant tumor foci (24). We previously synthesized the
major metabolite of equilenin, 4-hydroxyequilenin (4-
Ma ter ia ls a n d Meth od s
Ch em ica ls a n d Rea gen ts. Ca u tion : All catechol estrogens
were handled in accordance with NIH guidelines for the Labora-
tory Use of Chemical Carcinogens (29). All chemicals were
purchased from Aldrich (Milwaukee, WI), Fisher Scientific
(Itasca, IL), or Sigma (St. Louis, MO) unless stated otherwise.
4-OHEN was synthesized by treating equilin with Fremy’s salt
as described previously (26, 30) with minor modifications (27).
8-Oxo-dA was prepared as described previously (31). Briefly,
2′-deoxyadenosine (500 mg) was treated with 0.5 M sodium
acetate containing a saturated bromine/water solution, giving
8-bromo-2′-deoxyadenosine (40% yield): 1H NMR (DMSO-d6) δ
2.18-2.23 (m, 1H, 2′-H), 3.22-3.37 (m, 1H, 2′-H), 3.48-3.52 (m,
1H, 5′-H), 3.62-3.68 (m, 1H, 5′-H), 3.90 (m, 1H, 4′-H), 4.48 (m,
1H, 3′-H), 5.27-5.37 (m, 2H, 3′,5′-OH), 6.30 (m, 1H, 1′-H), 7.55
(s, 2H, 6-NH2), 8.11 (s, 1H, 2-H); 13C NMR (DMSO-d6) δ 37.8,
62.9, 72.0, 87.2, 89.1, 120.5, 127.5, 150.7, 153.2, 155.9; UV (CH3-
OH) 214, 264 nm; electrospray MS (positive ion, relative
intensity) m/z 352 (MH+, 100%), 354 (M + 2 + H+, 100%).
8-Bromo-2′-deoxyadenosine was converted to 8-oxo-dA as fol-
lows. DMSO (15 mL), benzyl alcohol (5 mL), and sodium metal
(170 mg, 7.39 mmol) were combined, and the solution was
stirred until the sodium dissolved. 8-Bromo-2′-deoxyadenosine
was added to the solution, and the mixture was stirred for 26 h
at 70 °C. The reaction was neutralized with glacial acetic acid,
and the solid was precipitated with 450 mL of diethyl ether.
After filtration, the precipitate was washed with diethyl ether
and methanol and then purified by flash chromatography with
CH3OH/CHCl3/acetone (1:5:1) as the eluant. Removal of solvent
en vacuo gave 8-oxo-2′-deoxyadenosine (28% yield): 1H NMR
(DMSO-d6) δ 2.00 (m, 1H, 2′-H), 2.96 (m, 1H, 2′-H), 3.45 (m,
1H, 5′-H), 3.59 (m, 1H, 5′-H), 3.81 (s, 1H, 4′-H), 4.38 (s, 1H, 3′-
F igu r e 2. Proposed model for 4-OHEN toxicity and DNA damage.

Catechol Estrogen Induced DNA Damage
Chem. Res. Toxicol., Vol. 11, No. 9, 1998 1107
H), 5.14-5.21 (m, 2H, 3′,5′-OH), 6.11-6.36 (m, 1H, 1′-H), 6.64
(s, 2H, 6-NH2), 8.01 (s, 1H, 2-H), 10.46 (broad s, 1H, 8-OH); 13
C
NMR (DMSO-d6) δ 37.7, 63.3, 72.7, 83.3, 88.5, 104.3, 146.8,
147.9, 150.8, 152.6; UV (CH3OH) 210, 270 nm; electrospray MS
(positive ion, relative intensity) m/z 268 (MH+, 100%); electro-
spray MS/MS (positive ion, relative intensity) of the m/z 268
ion 152 (M - 116, 100%), 135 (20%), 125 (30%), 108 (20%),
similar to what has been reported in the literature (32).
DNA Sin gle-Str a n d Br ea k s P r od u ced by 4-OHEN. Typi-
cal reaction conditions were as follows. DNA [λ phage (Sigma),
0.5 µg/µL] was incubated with 4-OHEN (0.5 mM) in 50 mM
potassium phosphate buffer (10 µL) at pH 7.4 and 20 °C. In all
cases, the appropriate controls were included in the same
experiment. The solutions were mixed and incubated for 30
min. The DNA was denatured in boiling water for 3 min in the
presence of 5 µL of DNA sequencing stop solution (10 mM
NaOH, 95% formamide, 0.05% bromophenol blue, and 0.05%
xylene cyanol). The denatured DNA was immediately subjected
to electrophoresis on a 0.7% agarose gel with 0.5 µg/mL ethidium
bromide in 40 mM TRIS-acetate buffer containing 1 mM EDTA.
The DNA was visualized under UV irradiation at 312 nm. After
electrophoresis for 1 h, the agarose gel was photographed with
a Polaroid 3000 camera.
F igu r e 3. Dose dependence of 4-OHEN-mediated DNA dam-
age. (A) Agarose gel electrophoresis of λ DNA (0.5 µg/µL)
incubated at 20 °C with various concentrations of 4-OHEN: lane
1, molecular weight markers (1-12 kb); lane 2, 0.4 µL of acetone;
lane 3, 4-OHEN (0.1 mM); lane 4, 4-OHEN (0.5 mM); and lane
5, 4-OHEN (2 mM). (B) As in panel A using DNA which had
not been denatured.
described previously (5). The deoxynucleosides were separated
as described above using a HPLC apparatus (Shimadzu LC-
10AS) coupled to UV photodiode array and electrochemical (EC)
detectors (ESA CoulArray). For EC detection, the channels,
numbered 1-4, were set at the following values: 200, 290, 380,
and 430 mV, respectively. Aliquots (50 mL) of the DNA
hydrolysate were injected onto a Partisil 5 µm ODS-3 reverse
phase analytical column [4.6 mm × 250 mm (i.d.); Whatman,
Clifton, NJ ] that was maintained at 25 °C. The mobile phase
contained 4 mM citric acid, 8 mM ammonium acetate, 10%
methanol, and 20 mg/L EDTA, saturated with helium (ultrapure
grade), at pH 4 with a flow rate of 1 mL/min. The 8-oxo-dG
was eluted with a retention time of 27.5 min. Quantification
of 8-oxo-dG was obtained from EC channel 2 (290 mV) analysis.
Quantitations of dC, dT, dG, and dA were performed by UV
absorption and monitored at 254 nm, and they were eluted with
retention times of 9.6, 18, 19.9, and 30.5 min, respectively.
Concentrations of 8-oxo-dG were expressed relative to the
concentrations of dG detected by UV absorbance at 254 nm. A
Student’s t test was used for statistical analysis of the data.
In str u m en ta tion . HPLC experiments were performed on
the above-mentioned Shimadzu LC-10A gradient HPLC system.
Peaks were integrated with Shimadzu EZ-Chrom software and
a 486-33 computer. UV spectra were measured with a Hewlett-
Packard model 8452 photodiode array UV spectrophotometer,
It was observed that an excess of GSH (10-fold relative to
the 4-OHEN concentration) could completely protect λ phage
DNA from 4-OHEN-mediated damage. As a result, we exploited
this effect to determine the time required for 4-OHEN to cause
DNA single-strand breaks. 4-OHEN (2 mM) was mixed with λ
phage DNA (0.5 µg/µL) in 80 µL of 50 mM potassium phosphate
buffer (pH 7.4) at 20 °C. At various time points, aliquots (10
µL) were removed and combined with 10 mM GSH. After 30
min, the aliquots were denatured and subjected to electrophore-
sis on a 0.7% agarose gel as described above.
4-OHEN-Med ia ted Oxid a tion of DNA Ba ses. Calf thymus
DNA (Sigma) (50 mg) was dissolved in 10 mL of TE buffer [10
mM Tris, 5 mM NaCl, and 1 mM EDTA (pH 7.8)] and repeatedly
precipitated with 70% ethanol/water to remove all impurities.
A 1 mL solution of DNA (2 mg/mL) in 50 mM phosphate buffer
(pH 7.4) containing 1 mM EDTA was incubated with 4-OHEN
(2.0 mM) and ampicillin (100 µg/mL) for 3 h at 37 °C. At the
conclusion of the incubation, the DNA was precipitated with
70% ethanol/water and washed with three 5 mL portions of 70%
ethanol. The precipitated DNA was hydrolyzed to deoxynucleo-
sides using the following literature procedures with minor
modifications (33, 34). Briefly, the DNA was redissolved in 1
mL of DNase buffer [50 mM sodium acetate, 2 mM CaCl2, and
10 mM MgCl2 (pH 6.5)] and incubated with DNase I (20 units/
mg of DNA) for 20 h at 37 °C. Then 50 µL of 0.5 M Tris buffer
(pH 9) and snake venom phosphodiesterase (0.013 unit/mg of
DNA) were added, and the mixture was incubated at 37 °C for
4 h. This treatment resulted in complete hydrolysis to the
deoxynucleoside level. The DNA hydrolysates were neutralized
with 1 M HCl, and aliquots (80 µL) were analyzed by HPLC
with an Ultrasphere ODS column (4.6 mm × 250 mm, Beckman)
with a flow rate of 1.0 mL/min on a Shimadzu LC-10A gradient
HPLC apparatus equipped with a SIL-10A auto injector, a SPD-
M10AV UV/VIS photodiode array detector, and a SPD-10AV UV
detector set at 280 nm. The HPLC mobile phase consisted of
1.0% CH3OH in water which was increased to 5.0% CH3OH in
10 min, to 15% CH3OH in another 24 min, and to 90% CH3OH
in the last 6 min of the run. For LC/MS analysis (electrospray,
positive ion), aliquots (25 µL) were analyzed directly by HPLC
on a Hewlett-Packard (Palo Alto, CA) 1090L gradient HPLC
apparatus equipped with a photodiode array UV/vis absorbance
detector set at 230-350 nm and a 5989B MS Engine quadrupole
mass spectrometer. Under these conditions, the retention times
and MH+ ions of the oxidized deoxynucleosides were as fol-
lows: 8-oxo-dG, 26 min, MH+, 284; and 8-oxo-dA, 37 min, MH+,
268.
1
and H NMR and 13C NMR spectra were obtained with a Bruker
Avance 300 spectrometer. Positive ion electrospray mass
spectra were obtained using a Hewlett-Packard 5989B MS
Engine quadrupole mass spectrometer equipped with a Chem-
Station data system and a high-flow pneumatic nebulizer-
assisted electrospray LC/MS interface. The mass spectrometer
was interfaced to the above-mentioned Hewlett-Packard gradi-
ent HPLC system. The quadrupole analyzer was maintained
at 120 °C, and unit resolution was used for all measurements.
Nitrogen at a pressure of 80 psi was used for nebulization of
the HPLC effluent, and nitrogen bath gas at 250 °C with a flow
rate of 10 L/min was used for evaporation of the solvent from
the electrospray system. The m/z range of 200-900 was
scanned every 2 s during LC/MS. LC/MS/MS experiments were
performed on
a Micromass quattro II electrospray triple-
quadrupole mass spectrometer. Samples were infused using a
syringe pump in 50% methanol/water containing 1% acetic acid.
Resu lts
Figure 3 shows the effects of incubating increasing
concentrations of 4-OHEN with λ phage DNA. The
damage is characteristic of single-strand breaks since no
fragmentation was observed in similar experiments with
double-stranded DNA which had not been denatured
8-Oxo-d G An a lysis. Deoxyguanosine (dG) and 8-oxo-dG
concentrations in the DNA hydrolysates were determined by
HPLC with UV and electrochemical detection, respectively, as

1108 Chem. Res. Toxicol., Vol. 11, No. 9, 1998
Chen et al.
F igu r e 5. Effect of redox cycling on 4-OHEN-mediated DNA
damage. Agarose gel electrophoresis of λ DNA (0.5 µg/µL)
incubated at 20 °C with various combinations of 4-OHEN,
NADH, and/or CuCl2: lane 1, molecular weight markers (1-12
kb); lane 2, 0.4 µL of acetone; lane 3, 4-OHEN (0.5 mM); lane 4,
4-OHEN (0.5 mM) and NADH (4 mM); lane 5, 4-OHEN (0.5
mM), NADH (4 mM), and CuCl2 (0.1 mM); lane 6, NADH (4
mM) and CuCl2 (0.1 mM); lane 7, NADH (4 mM); and lane 8,
NADH (4 mM) alone.
F igu r e 4. Time course for 4-OHEN-mediated DNA damage.
Agarose gel electrophoresis of λ DNA (0.5 µg/µL) incubated at
20 °C with 4-OHEN (1 mM). GSH (10 mM) was added at various
times to quench 4-OHEN and prevent further DNA damage:
lane 1, molecular weight markers (1-12 kb); lane 2, 0 min; lane
3, 1 min; lane 4, 3 min; lane 5, 6 min; lane 6, 10 min; lane 7, 20
min; and lane 8, 30 min.
prior to electrophoresis (Figure 3B). DNA in the vehicle-
treated sample (Figure 3A, lane 2) is clearly visible,
whereas increasing concentrations of 4-OHEN caused a
dose-dependent disappearance of high-molecular weight
(MW) DNA and an enhancement in DNA with fewer
kilobases indicative of strand scission.
and CuCl2, or NADH alone, had no effect on DNA as
shown in lanes 6 and 7, respectively. NADH does appear
as a fluorescent spot in these control samples but not in
lanes 3-5 since it is completely consumed by continuous
reduction of 4-OHEN-o-quinone (25).
The ability of scavengers of reactive oxygen species to
protect DNA from 4-OHEN-mediated damage is shown
in Figure 6. Including azide (lane 4), mannitol (lane 5),
and catalase (lane 6) which scavenge singlet oxygen, free
hydroxyl radicals, and hydrogen peroxide respectively,
completely protected DNA from single-strand breaks. In
contrast, SOD could not protect the DNA from 4-OHEN.
These data strongly suggest that singlet oxygen, hydroxyl
radicals, and hydrogen peroxide play a major role in
4-OHEN-mediated DNA damage.
Determining the type of lesions on DNA bases caused
by redox cycling of 4-OHEN was of interest. When calf
thymus DNA was treated with 4-OHEN, hydrolyzed to
deoxynucleosides, and analyzed by HPLC, chromato-
grams similar to the one in Figure 7B were obtained.
Very significant increases in 8-oxo-dG levels were ob-
served relative to that of the vehicle-treated control, i.e.,
100-fold greater for the 4-OHEN-treated sample com-
pared to the control (Table 1). Including NADH in the
incubation further increased the 8-oxo-dG peak (Figure
7C and Table 1); however, additional oxidized deoxy-
nucleosides were also observed, including 8-oxo-dA as
well as some unidentified peaks. The identity of 8-oxo-
dA was confirmed by co-injection of the synthesized
standard and comparisons of LC/MS and UV analyses.
By constructing a standard curve with the synthesized
8-oxo-dA, we have determined the amount of 8-oxo-dA
formed to be 0.094 ( 0.008 8-oxo-dA/dA% which is
The effect of incubation time on the ability of 4-OHEN
to damage DNA is shown in Figure 4. In this experi-
ment, we took advantage of the observation that high
concentrations of GSH can completely protect DNA from
4-OHEN-mediated damage since GSH can trap the
4-OHEN-o-quinone (J . L. Bolton et al., unpublished
results). Although the resulting GSH conjugates also
have the potential to redox cycle (35, 36), the high
concentration of GSH is much more effective at scaveng-
ing reactive oxygen species as compared to DNA and
DNA remains intact. By varying the time of addition of
GSH, we can determine the time course of 4-OHEN
single-strand cleavage. Figure 4 shows that within 1 min
of reaction with 4-OHEN a significant increase in DNA
damage has occurred (Figure 4, lane 3). The extent of
damage increases with time until the 30 min time point,
where no intact DNA can be detected (Figure 4, lane 8).
To investigate the effects of 4-OHEN-mediated redox
cycling, we incubated 4-OHEN with λ phage DNA and
NADH (Figure 5, lane 4). Increases in the amount of
single-strand cleavage were observed relative to that for
4-OHEN alone (Figure 5, lane 3) or vehicle-treated DNA
(Figure 5, lane 2). The addition of CuCl2 (Figure 5, lane
5) almost completely destroyed the DNA likely due to
formation of copper hydroperoxide complexes (3, 4, 37)
which could result from reaction of copper with hydrogen
peroxide generated by redox cycling of 4-OHEN. NADH

Catechol Estrogen Induced DNA Damage
Chem. Res. Toxicol., Vol. 11, No. 9, 1998 1109
modify DNA (27, 28). The electrophoretic mobility of
double-stranded DNA was unaffected under similar
reaction conditions which suggests that 4-OHEN causes
single-strand breaks in DNA. Similarly, when 4-hydroxy-
estrone-o-quinone was incubated in MCF-7 cells, single-
strand breaks in cellular DNA were also formed but
double-strand DNA breaks were not detected (12). The
damage is dose- (Figure 3) and time-dependent (Figure
4), could be increased in the presence of reducing agents,
and reducing agents and CuCl2 (Figure 5), and could be
prevented in the presence of scavengers of singlet oxygen,
hydroxyl radicals, and hydrogen peroxide (Figure 6).
Copper is closely associated with DNA in the cell,
particularly with guanine-rich regions (38). It has been
reported that copper interacts with hydrogen peroxide,
forming copper peroxide complexes as shown below (3,
4), which are likely involved in the enhanced DNA
damage observed in this study (adapted from ref 3).
DNA-(CuII)2 + H2O2 f DNA-(CuI)2 + O2
DNA-CuI + H2O2 f DNA-CuIOOH
The ability of copper to catalyze DNA damage has been
previously reported for other hydroxylated aromatic
compounds, including the endogenous catechol estrogens
(39), hydroquinone (37), and the catechol metabolite of
the antitumor agent etoposide (40). In addition, a recent
report has shown that o-quinones formed from polycyclic
aromatic hydrocarbon cause a significant amount of DNA
strand scission in the presence of NADPH and CuCl2 (41)
which suggests that similarities in structure between
4-OHEN-o-quinone and these PAH metabolites may
explain their proposed comparable cytotoxic and mu-
tagenic mechanisms (42).
Treating calf thymus DNA with 4-OHEN, hydrolyzing
the DNA to deoxynucleosides, and HPLC analysis re-
vealed a substantial increase in 8-oxo-dG formation (100-
fold). Supporting this, previous reports have shown that
incubations with 4-OHEN-o-quinone, DNA, and hamster
liver microsomes also enhanced 8-oxo-dG formation 2-fold
relative to control samples (26). We believe that the
substantial increases in 8-oxo-dG levels observed in this
study are the result of starting from the catechol rather
than from the o-quinone, since autoxidation of the
catechol generates reactive oxygen species leading to
8-oxo-dG formation. Interestingly, when we included
NADH in the incubations with 4-OHEN and DNA, 8-oxo-
dA was observed (Figure 7C). These data are particu-
larly intriguing since 8-oxo-A (43) and 8-oxo-G (44) are
mutagenic lesions associated with a heightened cancer
risk. These and other data are evidence for a mechanism
of estrogen-induced tumor initiation by redox cycling of
estrogen metabolites generating reactive oxygen species
which damage DNA as shown in Figure 2.
F igu r e 6. Effect of scavengers of ROS on 4-OHEN-mediated
DNA damage. Agarose gel electrophoresis of λ DNA (0.5 µg/µL)
incubated at 20 °C with various combinations of 4-OHEN and
Scavengers of ROS: lane 1, molecular weight markers (1-12
kb); lane 2, 0.4 µL of acetone; lane 3, 4-OHEN (2 mM); lane 4,
4-OHEN (2 mM) and azide (50 mM); lane 5, 4-OHEN (2 mM)
and mannitol (50 mM); lane 6, 4-OHEN (2 mM) and catalase (5
units); and lane 7, 4-OHEN (2 mM) and SOD (5 units).
Ta ble 1. 4-OHEN-Med ia ted 8-Oxid a tion of Gu a n in e Ba ses
of DNAa
reaction conditions
8-oxo-dG/dG%
acetone (40 µL/mL)
NADH
NADH and CuCl2
4-OHEN
4-OHEN and NADH
4-OHEN, NADH, and CuCl2
0.005 ( 0.001b
0.003 ( 0
0.067 ( 0.005
2.9 ( 0.4
3.7 ( 0.2c
3.8 ( 0.5c
a
Calf thymus DNA (2 mg/mL) was treated with 4-OHEN (2
mM) in phosphate buffer at pH 7.4 and 37 °C for 3 h. The DNA
was precipitated, washed with ethanol, and hydrolyzed to deoxy-
nucleosides. The hydrolysate was analyzed and its 8-OHdG content
determined as described in Materials and Methods. The concen-
tration of NADH was 3 mM, and that of CuCl2 was 0.1 mM. The
data represent an average ( SD of three determinations. b Average
of two determinations. c Significantly different from that for
4-OHEN alone (p < 0.05).
considerably less than the amount of 8-oxo-dG produced
(Table 1). The peak at 30 min was determined to be
adenosine by comparison of the MS/MS daughter ion
spectra with that of the authentic standard. The source
of adenosine is unknown at present; however, it may
come from degradation of NADH by copper peroxy
radicals.
We previously showed that 4-OHEN has the ability to
form highly unusual cyclic adducts with deoxynucleosides
and DNA (27, 28). Three different types of adducts were
formed, including unstable adenine adducts which readily
depurinate, leaving apurinic sites on the DNA. Stable
bulky adducts were also produced with guanine and
cytosine which if formed in vivo must be repaired by
nucleotide excision repair enzymes. In this investigation,
we have shown that 4-OHEN also has the ability to
oxidize DNA bases, forming a number of different mu-
tagenic lesions. Neither DNA alkylation nor oxidation
Discu ssion
Our data clearly demonstrate that 4-OHEN is capable
of causing DNA single-strand breaks and oxidative
damage to DNA bases in addition to our previous reports
which showed that 4-OHEN has the ability to covalently

1110 Chem. Res. Toxicol., Vol. 11, No. 9, 1998
Chen et al.
intermediates formed from estrogen replacement formu-
lations and their cellular targets be thoroughly explored.
Ack n ow led gm en t. We thank Dr. Alexander Ney-
fakh for access to the electrophoresis apparatus and for
helpful discussions. This research was supported by NIH
Grants CA73638 (J.L.B.), GM39338 and ES07784 (S.S.L.),
and CA77071 (R.B.v.B.).
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