ADVANCES IN CLINICAL CHEMISTRY, VOL.

38

ESTROGEN METABOLITES, CONJUGATES, AND DNA

ADDUCTS: POSSIBLE BIOMARKERS FOR RISK OF
BREAST, PROSTATE, AND OTHER HUMAN CANCERS

Eleanor G. Rogan and Ercole L. Cavalieri

Eppley Institute for Research in Cancer and Allied Diseases,

University of Nebraska Medical Center,
Omaha, Nebraska 68198

1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 135
1.1. Mechanisms of Tumor Initiation by Estrogens . . . . . . . . . . . . . . . . . . . . . . . . . . 135
1.2. Metabolism of Estrogens. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 136
1.3. Estrogens as Possible Biomarkers for Risk of Developing Cancer . . . . . . . . . 138
2. Analysis of Estrogens and Their Metabolites, Conjugates, and
Depurinating DNA Adducts. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 139
2.1. Analysis of Estrogens. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 139
2.2. Analysis of Catechol Estrogens . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 141
2.3. Analysis of Estrogen Metabolites and Conjugates . . . . . . . . . . . . . . . . . . . . . . . 141
2.4. Analysis of Depurinating Catechol Estrogen-DNA Adducts . . . . . . . . . . . . . . 143
3. Biomarkers for Increased Risk of Developing Estrogen-Initiated Cancer . . . . . . . 144
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 146

1. Introduction

1.1. MECHANISMS OF TUMOR INITIATION BY ESTROGENS

Various types of evidence have implicated estrogens in the etiology of
human breast cancer (C5, C9, C10, F1, L5, L6). They are generally thought
to cause proliferation of breast epithelial cells through estrogen receptor-
mediated processes (F1). Rapidly proliferating cells are susceptible to genetic
errors during DNA replication, which, if uncorrected, can ultimately lead to
malignancy. While receptor-mediated processes may play an important role
in the development and growth of tumors, accumulating evidence suggests

135

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136 ROGAN AND CAVALIERI

that specific oxidative metabolites of estrogens, if formed, can be endogenous

ultimate carcinogens that react with DNA to cause the mutations leading
to initiation of cancer (C5, C9, C11). Thus, estrogen metabolites, conjugates,
and DNA adducts could serve as biomarkers for increased susceptibility to
breast, prostate, and other human cancers.
One hypothesis on the etiology of breast cancer has been that its induction
is caused by a covalent bond of 16-hydroxyestrone (16-OHE1), a metabo-
lite of E1, with the estradiol (E2) receptor. This receptor modification would
result in permanent, uncontrolled stimulation of cell proliferation by recep-
tor-mediated processes (B7, S1, S6). This hypothesis implies a correlation of
high levels of 16-OHE1 with induction of breast cancer. Over the years,
however, this hypothesis has never been substantiated.
Several lines of evidence, including metabolism and carcinogenicity
studies by Liehr and coworkers, led to the recognition that the 4-hydroxylated
estrogens play a major role in the genotoxic properties of estrogens (L5–L7).
We have hypothesized that the estrogens E1 and E2 initiate breast cancer by
reaction of their electrophilic metabolites, catechol estrogen-3,4-quinones
[E1(E2)-3,4-Q], with DNA to form depurinating adducts (C5, C9, C10).
These adducts generate apurinic sites, leading to mutations that may initiate
breast, prostate, and other human cancers (C5, C9, C11).

1.2. METABOLISM OF ESTROGENS

The estrogens E1 and E2 are obtained via aromatization of 4-androstene-3,
17-dione and testosterone, respectively, catalyzed by cytochrome P450(CYP)
19, aromatase (Fig. 1). E1 and E2, which are biochemically interconvertible
by the enzyme 17-estradiol dehydrogenase, are metabolized to the 2-cate-
chol estrogens, 2-OHE1(E2), and 4-OHE1(E2), predominantly catalyzed by
the activating enzymes CYP1A1 (S4) and 1B1 (H2, S2–S4), respectively, in
extrahepatic tissues. The estrogens are also metabolized, to a lesser extent, by
16-hydroxylation (not shown). The catechol estrogens are further oxidized
to the catechol estrogen quinones, E1(E2)-2,3-Q and E1(E2)-3,4-Q (Fig. 1). In
general, the catechol estrogens are inactivated by conjugating reactions, such
as glucuronidation and sulfation. A common pathway of inactivation in
extrahepatic tissues, however, occurs by O-methylation catalyzed by the
ubiquitous catechol-O-methyltransferase (COMT) (B1). If formation of E1
or E2 is excessive, due to overexpression of aromatase and/or the presence of
excess sulfatase that converts the stored E1 sulfate to E1, increased formation
of catechol estrogens is expected. In particular, the presence and/or induction
of CYP1B1 and other 4-hydroxylases could render the 4-OHE1(E2), which are
usually minor metabolites, as the major metabolites. Thus, conjugation of 4-
OHE1(E2) via methylation in extrahepatic tissues might become insuYcient,
FIG. 1. Formation, metabolism, conjugation, and DNA adducts of estrogens.
138 ROGAN AND CAVALIERI

and competitive catalytic oxidation of 4-OHE1(E2) to E1(E2)-3,4-Q could

occur (Fig. 1).
Protection at the quinone level can occur by conjugation of E1(E2)-Q with
glutathione (GSH), catalyzed by S-transferases (Fig. 1). A second inactivat-
ing process for E1(E2)-Q is their reduction to catechol estrogens by quinone
reductase. If these two inactivating processes are not eVective, E1(E2)-Q may
react with DNA to form stable and depurinating adducts (C5, C8–C10, D5,
L2, S5). We hypothesize that imbalances in estrogen homeostasis, that is, the
equilibrium between activating and protective enzymes with the scope of
avoiding formation of catechol estrogen semiquinones and quinones, can
lead to initiation of cancer by estrogens.

1.3. ESTROGENS AS POSSIBLE BIOMARKERS FOR

RISK OF DEVELOPING CANCER
Based on these considerations, the identification and quantification of
estrogen metabolites, conjugates, and depurinating DNA adducts in
human specimens could provide early diagnostic tools for determining the
risk of developing breast, prostate, and other human cancers. These possible
biomarkers could include the estrogens E1 and E2 themselves, the catechol
estrogens, methoxy catechol estrogens, catechol estrogen-GSH conjugates
and/or their derivatives, catechol estrogen-cysteine (Cys) and catechol estro-
gen-N-acetylcysteine (NAcCys) conjugates, as well as the depurinating DNA
adducts 4-OHE1(E2)-1-N3adenine (Ade), 4-OHE1(E2)-1-N7guanine (Gua),
and 2-OHE1(E2)-6-N3Ade (Fig. 2). The CE-GSH conjugates generally
undergo further metabolism by the mercapturic acid synthesis pathway
to generate catechol estrogen-Cys and, subsequently, catechol estrogen-
NAcCys conjugates (Fig. 3) (B6).

FIG. 2. Depurinating DNA adducts, 4-OHE1(E2)-1-N3Ade, 4-OHE1(E2)-1-N7Gua, and

2-OHE1(E2)-6-N3Ade.
 ESTROGEN BIOMARKERS FOR CANCER RISK 139

FIG. 3. GSH conjugation with the electrophilic compound RX, followed by mercapturic acid
biosynthesis to yield the NAcCys conjugate as the final product.

2. Analysis of Estrogens and Their Metabolites,

Conjugates, and Depurinating DNA Adducts

2.1. ANALYSIS OF ESTROGENS

Investigators have measured the levels of estrogens in women with and
without breast cancer since the 1970s (C1, D1). During the 1990s, estrogens
in human blood and tissues have been analyzed in a variety of ways. Some of
these analyses have been conducted on untreated blood serum samples (B3,
D4, K1, T4), but others have purified a fraction containing the estrogens,
usually by organic extraction of serum, followed by column chromatography
(C4, H1, H3, T2). Radioimmunoassays have been conducted to analyze E1,
E2, and other estrogens in both untreated and purified serum samples. In nine
diVerent studies (B2, B3, C4, D4, H1, H3, K1, T2, T4) that were combined
for meta-analysis (T1), the level of E1 in serum from women with and without
breast carcinoma ranged from approximately 50 to 150 fmol/ml and the level
of E2, from 20 to 150 fmol/ml. The relative risk of breast cancer for women
whose E2 levels were in the top quintile was twice that of women whose E2
levels were in the bottom quintile.
Since 2000, the estrogens E1 and E2 have been analyzed in tissues from a
variety of laboratory animal models. The estrogens were extracted from the
tissues and analyzed by HPLC with multichannel electrochemical detection
 TABLE 1
ANALYSIS OF ESTROGEN METABOLITES AND CONJUGATES IN HUMAN BREAST TISSUE FROM
WOMEN WITH AND WITHOUT BREAST CANCER

pmol/g tissuea

2-Methoxy- 4-Methoxy- Quinone

Breast tissue E1(E2) 2-OH-E1(E2) 4-OH-E1(E2) 16-OHE1(E2) E1(E2) E1(E2) Conjugatesb

Controls – 4.1
3.0 (43) 5.4
5.1 (24) 3.4
2.7 (10) 2.8
1.2 (33) 3.5
2.8 (16) 4.1
2.6 (27) 2.6
1.5 (29)
noncancer
subjects (49)
Breast cancer 8.0
6.8 (46) 4.5
4.9 (46) 13.3
13.2 (54) 3.5
2.7 (18) 1.9
1.1 (29) 3.2
2.4 (39) 8.2
7.0 (57)
cases (28)
pc n.s. n.s. 0.01 n.s. n.s. n.s. 0.003

Number in parentheses presents the percentage of positive samples (i.e., frequency of detection, %).
n.s. ¼ Statistically nonsignificant diVerences from controls.
a
Values are mean
S.D. of the positive samples.
b
Quinone conjugates are 4-OHE1(E2)-2-NAcCys, 4-OHE1(E2)-2-Cys, 2-OHE1(E2)-(1+4)-NAcCys, and 2-OHE1(E2)-(1+4)-Cys.
c
Statistically significant diVerences (compared to controls) were determined using the Wilcoxon rank sum test.
 ESTROGEN BIOMARKERS FOR CANCER RISK 141

(C7, C8, D2, L2). This methodology was also used to analyze the levels in
human nontumor breast tissue. In women without breast cancer, E1 and E2
combined together equaled 4 pmol/g, whereas the tissue from women with
breast carcinoma contained 8 pmol/g (Table 1) (R1). These tissue levels are
one to two orders of magnitude higher than the levels in blood.

2.2. ANALYSIS OF CATECHOL ESTROGENS

The catechol estrogens, 2-OHE1(E2) and 4-OHE1(E2), have been analyzed
in a variety of samples, along with the hydroxylated estrogen 16-OHE1.
These hydroxylated estrogens were first considered ‘‘unusual’’ estrogens and
were found at elevated levels in urine samples from women with breast
cancer (C1). The catechol estrogens and 16-OHE1 were analyzed as the
trimethylsilylethers in human breast tissue and breast cyst fluid by using gas
chromatography/mass spectrometry (GC/MS) after initial purification by
reversed-phase HPLC (C2). The catechol estrogens were analyzed in the
rat brain following a more complex procedure that involved extraction,
formation of the acetate derivatives, and several purification steps before
analysis by liquid chromatography-atmospheric pressure chemical ioniza-
tion-ion trap tandem mass spectrometry (MS/MS) (M1). In a study in 2002,
the catechol estrogens plus 16-OHE1 were analyzed in normal and tumor
tissue from human breast by using GC/MS coupled with HPLC (C3).
In addition, catechol estrogens, 16-OHE1, and methoxy catechol estro-
gens have been determined in nontumor breast tissue from women with and
without breast cancer (R1) and in selected tissues of laboratory animals (C7,
C8, D2, L2) by using HPLC coupled with multichannel electrochemical
detection (Fig. 4). The level of 4-OHE1(E2) was significantly higher in breast
tissue from women with breast carcinoma than in tissue from women without
breast cancer (Table 1) (R1), and tissue from women with breast cancer
contained significantly more 4-OHE1(E2) than 2-OHE1(E2), as seen previ-
ously in much smaller studies (C2, L4). The level of 16-OHE1 was about the
same in breast tissue from women with and without breast cancer (Table 1),
but slightly more methoxy catechol estrogens than unmodified catechol
estrogens were found in breast tissue from women without breast cancer,
suggesting that methylation of catechol estrogens to protect them from
oxidation to E1(E2)-Q happens more often in women without breast cancer.

2.3. ANALYSIS OF ESTROGEN METABOLITES AND CONJUGATES

Estrogen metabolites and conjugates have been analyzed in tissues from
laboratory animals and in human breast tissue by using HPLC with multi-
channel electrochemical detection. A total of 31 compounds, including the
142 ROGAN AND CAVALIERI

FIG. 4. Multichannel electrochemical response from HPLC of standard mixture of estrogens,

estrogen metabolites, estrogen conjugates, and estrogen-DNA adducts. The peak numbers
correspond to the compounds as follows: (1) 2-OHE2-1-SG, (2) 2-OHE2-4-SG, (3) 4-OHE2-2-
SG, (4&9) 2-OHE2-1(&4)-Cys, (5) 2-OHE1-1(þ4)-SG, (6) 4-OHE2-1-N7Gua, (7) 4-OHE1-2-SG,
(8) 4-OHE2-2-Cys, (10) 4-OHE1-1-N7Gua, (11) 2-OHE2-1-NAcCys, (12) 16-OHE2, (13)
2-OHE2-4-NAcCys, (14) 4-OHE1-2-Cys, (15) 2-OHE1-1(þ4)-Cys, (16) 4-OHE2-2-NAcCys,
(17) 2-OHE1-1(þ4)NAcCys, (18) 4-OHE1-2-NAcCys, (19) 16-OHE1, (20) 4-OHE2, (21)
2-OHE2, (22) 2-OHE1, (23) 4-OHE1, (24) E2, (25) 4-OCH3E2, (26) 2-OCH3E2, (27) E1, (28)
4-OCH3E1, (29) 2-OH-3-OCH3E2, (30) 2-OCH3E1, (31) 2-OH-3-OCH3E1.

estrogens E1 and E2 themselves, the catechol estrogens and methoxy

catechol estrogens, and conjugates formed by reaction of E1(E2)-Q with
GSH have been detected in one HPLC run (Fig. 4). Chronic treatment of
male Syrian golden hamsters with 4-OHE2 induces kidney tumors (L1, L3).
When hamsters were intraperitoneally injected with 4-OHE2 and the kidney
tissue analyzed 1 to 24 h later, the GSH, Cys, and NAcCys conjugates of
4-OHE1 and 4-OHE2 were identified in the picomole range, with 4-OHE2-
2-Cys predominating (D3). Urine collected for 24 h following treatment of
hamsters with 4-OHE2 contained both the methoxy catechol estrogens that
arise from methylation of catechol estrogens and the Cys and NAcCys
conjugates derived from reaction of E1(E2)-Q with GSH (T3).
Treatment of hamsters with E2 also resulted in the formation of catechol
estrogens and methoxy catechol estrogens in both the liver and kidney within
4 h, with the liver containing higher levels of the compounds, especially
2-OHE1(E2) and 2-methoxyE1(E2) (C8). If the hamsters were injected with
L-buthionine (S, R) sulfoximine to deplete the cellular levels of GSH, fol-
lowed by E2 2.5 h later, the GSH conjugates were virtually nondetectable
in both the liver and kidney, but the kidney now had detectable levels of the
4-OHE1(E2)-1-N7Gua adducts (C8).
Prostate carcinomas arise in Noble rats several months after injection with
E2 and implantation with testosterone (B5). When Noble rats were treated
with 4-OHE2 or E2-3,4-Q for 90 min and their prostates removed, dissected,
 ESTROGEN BIOMARKERS FOR CANCER RISK 143

and analyzed, 4-OHE1(E2), 4-methoxyE1(E2), the GSH conjugates (or the

derivative Cys and NAcCys conjugates) were detected in the four lobes of
the prostate (C7). The levels of these compounds suggest that areas of the
prostate susceptible to induction of carcinoma have less protection of cate-
chol estrogens by COMT, GSH, and quinone reductase, favoring reaction of
E1(E2)-3,4-Q with DNA.
In addition to studies of laboratory animals treated with estrogens, ana-
lyses have been conducted on tissue from untreated animals susceptible
to mammary tumors. A novel model of breast cancer was established by
crossing mice carrying the Wnt-1 transgene (100% of females develop spon-
taneous mammary tumors) with the estrogen receptor- knock-out (ERKO)
mouse line (B4). Mammary tumors develop in these mice despite the lack of
functional estrogen receptor-. Extracts of hyperplastic mammary tissue
contained the 4-catechol estrogens, but not the 4-methoxy catechol estrogens
or the 2-catechol estrogens and 2-methoxy catechol estrogens (D2), which
typically predominate in normal tissue of laboratory animals and humans. In
addition, the 4-catechol estrogen-GSH conjugates and their hydrolytic con-
jugates with Cys or NAcCys were detected, demonstrating formation of
E1(E2)-3,4-Q in this tumor-prone tissue (D2).
Catechol estrogen-GSH conjugates and their hydrolytic Cys or NAcCys
conjugates were identified and quantified in breast tissue from women with
breast carcinoma at significantly higher levels than in breast tissue from
women without breast cancer (Table 1) (R1). This finding demonstrates
that the E1(E2)-Q are present in human breast tissue, suggesting that the
quinones may react with DNA to generate mutations leading to breast
cancer.

2.4. ANALYSIS OF DEPURINATING CATECHOL ESTROGEN-DNA ADDUCTS

The quinones E1(E2)-2,3-Q and E1(E2)-3,4-Q can react with DNA to form
very small amounts of stable adducts and larger amounts of six depurinating
adducts: 4-OHE1(E2)-1-N3Ade, 4-OHE1(E2)-1-N7Gua, and 2-OHE1(E2)-6-
N3Ade (Fig. 2) (C6, C10, L2, S5). The depurinating adducts derived from
reaction of E1(E2)-3,4-quinone are far more abundant than those coming
from E1(E2)-2,3-quinone (C6). The apurinic sites resulting from loss of the
depurinating Ade adducts have been hypothesized to generate the mutations
initiating cancer (C5, C9, C11). Thus, depurinating catechol estrogen-DNA
adducts are very promising biomarkers for susceptibility to estrogen-initiated
cancer.
When hamsters were intraperitoneally injected with 4-OHE2 and the kid-
ney tissue analyzed 1 to 24 h later, both the 4-OHE1-1-N7Gua and 4-OHE2-
1-N7Gua adducts were identified by mass spectrometry (D3). The N7Gua
144 ROGAN AND CAVALIERI

adducts were also detected by mass spectrometry in 24-h urine samples

collected from these hamsters (T3). As noted in Section 2.3, the 4-OHE1-1-
N7Gua and 4-OHE2-1-N7Gua adducts were not detectable in the hamster
kidney 2 h after injection of E2. The adducts were detected in the kidney by
HPLC coupled with electrochemical detection when the hamsters had been
pretreated with L-buthionine(S,R)sulfoximine to deplete GSH (C8).
Equimolar amounts of the DNA adducts 4-OHE2-1-N3Ade and 4-OHE2-
1-N7Gua (12 mol/mol DNA-P) have been found in the skin of female
SENCAR mice 4 h after topical treatment with E2-3,4-Q (C11). Higher
amounts of these adducts have been detected in the mammary glands of
female ACI rats following intramammillary injection of E2-3,4-Q (C12). In
both cases, the adducts were identified and quantified by HPLC coupled with
multichannel electrochemical detection.

3. Biomarkers for Increased Risk of

Developing Estrogen-Initiated Cancer

Many types of markers are being evaluated to estimate risk of developing

breast cancer. These include breast density; body mass index; expression of
BRCA1 and/or BRCA2 gene; cytological changes in breast epithelial cells
collected by ductal lavage; and levels of estrogens and androgens in serum,
breast tissue, or nipple aspirate fluid. Some studies suggest that levels of
selected estrogen metabolites, estrogen conjugates, and/or depurinating es-
trogen-DNA adducts in breast ductal fluid, collected either by nipple aspira-
tion or ductal lavage, could prove to be early biomarkers of susceptibility to
breast cancer. For example, in the recent study of estrogen metabolites and
conjugates in breast tissue, the levels of both 4-OHE1(E2) and the combined
4-catechol estrogen-GSH, Cys, and NAcCys conjugates were significantly
higher in women with breast carcinoma than in women without breast cancer
(Table 1) (R1). Breast fluid is particularly attractive as a source of biomar-
kers because the estrogen metabolites are more concentrated in breast fluid
(C2) and it can be collected by noninvasive means.
In the future, analysis of small molecules, such as estrogen metabolites,
estrogen conjugates, and depurinating estrogen-DNA adducts as biomarkers
can be accomplished by using liquid chromatography/mass spectrometry.
Especially promising is capillary HPLC through a short column, such as a
guard column, preceding MS/MS analysis. This approach is being used now
to analyze serum for a variety of small molecules, and it should also be very
eVective to analyze breast fluid. Estrogen metabolites with identical molecu-
lar weights, such as 2-OHE1(E2) and 4-OHE1(E2), can readily be distin-
guished by MS/MS (Fig. 5). Thus, if high levels of 4-OHE1(E2) turn out to
 ESTROGEN BIOMARKERS FOR CANCER RISK 145

FIG. 5. MS/MS of 2-OHE2 (top) and 4-OHE2 (bottom). The 2 catechol estrogens have the
same molecular weight, but are distinguishable by the primary daughter: fragment m/z ¼ 147 for
2-OHE2 and m/z ¼ 161 for 4-OHE2.

be a biomarker of elevated risk of breast cancer, as suggested by the data in

Table 1, these estrogen metabolites can be analyzed by known MS/MS
techniques. Similarly, the conjugates formed by reaction of E1(E2)-Q with
GSH (Table 1) may be found to be biomarkers of susceptibility to breast
cancer. The depurinating estrogen-DNA adducts, which are a direct mea-
sure of DNA damage, may ultimately be definitive biomarkers of risk of
146 ROGAN AND CAVALIERI

FIG. 6. LC/MS/MS of 4-OHE1(E2)-1-N7Gua and 4-OHE1(E2)-1-N3Ade. The adducts are

identified by the transition of parent ion to daughter ion after application of collision energy.

developing breast cancer. These adducts can be analyzed and distinguished

by using HPLC with MS detection (LC/MS) with a limit of detection of
approximately 100 femtomoles (Fig. 6).
Application of LC/MS/MS techniques coupled with minimal cleanup of
samples is a promising approach for analyzing biomarkers of breast cancer
susceptibility. The best biomarkers need to be identified, but a number of
estrogen metabolites, estrogen conjugates, and depurinating estrogen-DNA
adducts are likely candidates. Ongoing comparisons of women with and
without breast cancer will allow us to identify the most promising candidate
biomarkers of susceptibility. The most useful biomarkers will be validated
through prospective studies that follow development of breast cancer in
selected populations of women.

ACKNOWLEDGMENTS
We thank Dr. Sandra J. Gunselman for mass spectra. This research was supported by U.S.
Public Health Service grants P01 CA49210 and R01 CA49917 from the National Cancer
Institute. Core support in the Eppley Institute is provided by grant P30 CA36727 from the
National Cancer Institute.

REFERENCES
B1. Ball, P., and Knuppen, R., Catechol oestrogens (2- and 4-hydroxyestrogens):
Chemistry, biogenesis, metabolism, occurrence, and physiological significance. Acta
Endocrinol. (Copenhagen) 93(Suppl. 232), 1–127 (1980).
B2. Barrett-Connor, E., Friedlander, N. J., and Khaw, K. T., Dehydroepiandrosterone
sulfate and breast cancer risk. Cancer Res. 50, 6571–6574 (1990).
B3. Berrino, F., Muti, P., Micheli, A., et al., Serum sex hormone levels after menopause and
subsequent breast cancer. J. Natl. Cancer Inst. 88, 291–296 (1996).
B4. Bocchinfuso, W. P., Hively, W. P., Couse, J. F., Varmus, H. E., and Korach, E. S.,
A mouse mammary tumor virus-Wnt-1 transgene induces mammary gland hyperplasia
and tumorigenesis in mice lacking estrogen receptor. Cancer Res. 59, 1869–1876 (1999).
 ESTROGEN BIOMARKERS FOR CANCER RISK 147

B5. Bosland, M. C., Ford, H., and Horton, L., Induction at high incidence of ductal
prostate adenocarcinoma in NBL/Cr and Sprague-Dawley Hsd:SD rats treated with a
combination of testosterone and estradiol-17 or diethylstilbestrol. Carcinogenesis 16,
1311–1317 (1995).
B6. Boyland, E., and Chausseaud, L. F., The role of glutathione and glutathione
S-transferases in mercapturic acid biosynthesis. Adv. Enzymol. 32, 173–219 (1969).
B7. Bradlow, H. L., Hershcopf, R. J., Martucci, C. P., and Fishman, J., Estradiol
16-hydroxylation in the mouse correlates with mammary tumor incidence and presence
of murine mammary tumor virus. A possible model for the hormonal etiology of breast
cancer in humans. Proc. Natl. Acad. Sci. USA 82, 6295–6299 (1985).
C1. Castagnetta, L., D’Agostino, G., LoCasto, M., Traina, A., and Leake, R. E., Breast
cancer: A comparison of response to endocrine therapy and oestrogen excretion
patterns including unusual metabolites. Br. J. Cancer 44, 670–674 (1981).
C2. Castagnetta, L. A., Granata, O. M., Arcuri, F. P., Polito, L. M., Rosati, F., and
Cartoni, G. P., Gas chromatography/mass spectrometry of catechol estrogens. Steroids
57, 437–443 (1992).
C3. Castagnetta, L. A., Granata, O. M., Traina, A., et al., Tissue content of hydroxyestrogens
in relation to survival of breast cancer patients. Clin. Cancer Res. 8, 3146–3155 (2002).
C4. Cauley, J. A., Lucas, F. L., Kuller, L. H., Stone, K., Browner, W., and Cummings, S. R.,
Elevated serum estradiol and testosterone concentrations are associated with high risk
for breast cancer. Study of Osteoporotic Fractures Research Group. Ann. Intern. Med.
130(4 Pt. 1), 270–277 (1999).
C5. Cavalieri, E., Frenkel, K., Liehr, J. G., Rogan, E., and Roy, D., Estrogens as
endogenous genotoxic agents: DNA adducts and mutations. In ‘‘JNCI Monograph 27:
Estrogens as Endogenous Carcinogens in the Breast and Prostate’’ (E. Cavalieri and E.
Rogan, eds.), pp. 75–93. Oxford Press, 2000.
C6. Cavalieri, E., Kohli, E., Zahid, M., and Rogan, E., Greater reactivity of estradiol-
3,4-quinone vs estradiol-2,3-quinone with DNA in the formation of depurinating DNA
adducts. Proc. Amer. Assoc. Cancer Res. 44(2nd ed.), 180 (2003).
C7. Cavalieri, E. L., Devanesan, P., Bosland, M. C., Badawi, A. F., and Rogan, E. G.,
Catechol estrogen metabolites and conjugates in diVerent regions of the prostate of
Noble rats treated with 4-hydroxyestradiol: Implications for estrogen-induced initiation
of prostate cancer. Carcinogenesis 23, 329–333 (2002).
C8. Cavalieri, E. L., Kumar, S., Todorovic, R., Higginbotham, S., Badawi, A. F., and
Rogan, E. G., Imbalance of estrogen homeostasis in kidney and liver of hamsters
treated with estradiol: Implications for estrogen-induced initiation of renal tumors.
Chem. Res. Toxicol. 14, 1041–1050 (2001).
C9. Cavalieri, E. L., Rogan, E. G., and Chakravarti, D., Initiation of cancer and other
diseases by catechol ortho-quinones: A unifying mechanism. Cell & Mol. Life Sci. 59,
665–681 (2002).
C10. Cavalieri, E. L., Stack, D. E., Devanesan, P. D., et al., Molecular origin of cancer:
Catechol estrogen-3,4-quinones as endogenous tumor initiators. Proc. Natl. Acad. Sci.
USA 94, 10937–10942 (1997).
C11. Chakravarti, D., Mailander, P., Li, K.-M., et al., Evidence that a burst of DNA
depurination in SENCAR mouse skin induces error-prone repair and forms mutations
in the H-ras gene. Oncogene 20, 7945–7953 (2001).
C12. Chakravarti, D., Mailander, P. C., Higginbotham, S., Cavalieri, E. L., and Rogan,
E. G., The catechol estrogen-3,4-quinone metabolites induce mutations in the mammary
gland of ACI rats. Proc. Amer. Assoc. Cancer Res. 44(2nd ed.), 180 (2003).
148 ROGAN AND CAVALIERI

D1. Dao, T. L., Metabolism of estrogens in breast cancer. Biochim. Biophys. Acta 560,
397–426 (1979).
D2. Devanesan, P., Santen, R. J., Bocchinfuso, W. P., Korach, K. S., Rogan, E. G., and
Cavalieri, E. L., Catechol estrogen metabolites and conjugates in mammary tumors and
hyperplastic tissue from estrogen receptor-knock-out (ERKO)/Wnt-1 mice: Implica-
tions for initiation of mammary tumors. Carcinogenesis 22, 1573–1576 (2001).
D3. Devanesan, P., Todorovic, R., Zhao, J., Gross, M. L., Rogan, E. G., and Cavalieri,
E. L., Catechol estrogen conjugates and DNA adducts in the kidney of male Syrian
golden hamsters treated with 4-hydroxyestradiol: Potential biomarkers for estrogen-
initiated cancer. Carcinogenesis 22, 489–497 (2001).
D4. Dorgan, J. F., Longcope, C., Stephenson, H. E., Jr., et al., Relation of prediagnostic serum
estrogen and androgen levels to breast cancer risk. Cancer Epidemiol. Biomarkers Prev. 5,
533–539 (1996).
D5. Dwivedy, I., Devanesan, P., Cremonesi, P., Rogan, E., and Cavalieri, E., 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 (1992).
F1. Feigelson, H. S., and Henderson, B. E., Estrogens and breast cancer. Carcinogenesis 17,
2279–2284 (1996).
H1. Hankinson, S. E., Willett, W. C., Manson, J. E., et al., Plasma sex steroid hormone
levels and risk of breast cancer in postmenopausal women. J. Natl. Cancer Inst. 90,
1292–1299 (1998).
H2. Hayes, C. L., Spink, D. C., Spink, B. C., Cao, J. Q., Walker, N. J., and Sutter, T. R.,
17-estradiol hydroxylation catalyzed by human P450 1B1. Proc. Natl. Acad. Sci. USA
93, 9776–9781 (1996).
H3. Helzlsouer, K. J., Alberg, A. J., Bush, T. L., Longcope, C., Gordon, G. B., and
Comstock, G. W., A prospective study of endogenous hormones and breast cancer.
Cancer Detect. Prev. 18, 79–85 (1994).
K1. Kabuto, M., Akiba, S., Stevens, R. G., Nerishi, K., and Land, C. E., A prospective
study of estradiol and breast cancer in Japanese women. Cancer Epidemiol. Biomarkers
Prev. 9, 575–579 (2000).
L1. Li, J. J., and Li, S. A., Estrogen carcinogenesis in Syrian hamster tissue: Role of
metabolism. Fed. Proc. 46, 1858–1863 (1987).
L2. Li, K-M., Todorovic, R., Devanesan, P., et al., Metabolism and DNA binding studies of
4-hydroxyestradiol and estradiol-3,4-quinone in vitro and in female ACI rat mammary
gland in vivo. Carcinogenesis 25, 289–297 (2004).
L3. Liehr, J. G., Fang, W. F., Sirbasku, D. A., and Ari-Ulubelen, A., Carcinogenicity of
catecholestrogens in Syrian hamsters. J. Steroid Biochem. 24, 353–356 (1986).
L4. Liehr, J. G., and Ricci, M. J., 4-Hydroxylation of estrogens as marker of human
mammary tumors. Proc. Natl. Acad. Sci. USA 93, 3294–3296 (1996).
L5. Liehr, J. G., and Roy, D., Free radical generation by redox cycling of estrogens. Free
Rad. Biol. Med. 8, 415–423 (1990).
L6. Liehr, J. G., Genotoxic eVects of estrogens. Mutat. Res. 238, 269–276 (1990).
L7. Liehr, J. G., Is estradiol a genotoxic mutagenic carcinogen? Endocr. Rev. 21, 40–54 (2000).
M1. Mitamura, K., Yatera, M., and Shimada, K., Studies on neurosteroids. Part XIII.
Characterization of catechol estrogens in rat brains using liquid chromatography-mass
spectrometry-mass spectrometry. Analyst 125, 811–814 (2000).
R1. Rogan, E. G., Badawi, A. F., Devanesan, P. D., et al., Relative imbalances in estrogen
metabolism and conjugation in breast tissue of women with carcinoma: Potential
biomarkers of susceptibility to cancer. Carcinogenesis 24, 697–702 (2003).
 ESTROGEN BIOMARKERS FOR CANCER RISK 149

S1. Schneider, J., Kinne, D., Fracchia, A., et al., Abnormal oxidative metabolism of
estradiol in women with breast cancer. Proc. Natl. Acad. Sci. USA 79, 3047–3051 (1982).
S2. Spink, D. C., Hayes, C. L., Young, N. R., et al., The eVects of 2,3,7,8-tetrachlorodibenzo-
p-dioxin on estrogen metabolism in MCF-7 breast cancer cells: Evidence for induction of a
novel 17-estradiol 4-hydroxylase. J. Steroid Biochem. Mol. Biol. 51, 251–258 (1994).
S3. Spink, D. C., Spink, B. C., Cao, J. Q., et al., Induction of cytochrome P450 1B1 and
catechol estrogen metabolism in ACHN human renal adenocarcinoma cells. J. Steroid
Biochem. Mol. Biol. 62, 223–232 (1997).
S4. Spink, D. C., Spink, B. C., Cao, J. Q., et al., DiVerential expression of CYP1A1 and
CYP1B1 in human breast epithelial cells and breast tumor cells. Carcinogenesis 19,
291–298 (1998).
S5. Stack, D., Byun, J., Gross, M. L., Rogan, E. G., and Cavalieri, E., Molecular
characteristics of catechol estrogen quinones in reactions with deoxyribonucleosides.
Chem. Res. Toxicol. 9, 851–859 (1996).
S6. Swaneck, G. E., and Fishman, J., Covalent binding of the endogenous estrogen
16-hydroxyestrone to estradiol receptor in human breast cancer cells: Characterization
and intranuclear localization. Proc. Natl. Acad. Sci. USA 85, 7831–7835 (1988).
T1. The Endogenous Hormones and Breast Cancer Collaborative Group, Endogenous sex
hormones and breast cancer in postmenopausal women: Reanalysis of nine prospective
studies. J. Natl. Cancer Inst. 94, 606–616 (2002).
T2. Thomas, H. V., Key, T. J., Allen, D. S., et al., A prospective study of endogenous serum
hormone concentrations and breast cancer risk in post-menopausal women on the
island of Guernsey. Br. J. Cancer 76, 401–405 (1997).
T3. Todorovic, R., Devanesan, P., Higginbotham, S., et al., Analysis of potential
biomarkers of estrogen-initiated cancer in the urine of Syrian golden hamsters treated
with 4-hydroxyestradiol. Carcinogenesis 22, 905–911 (2001).
T4. Toniolo, P. G., Levitz, M., Zeleniuch-Jacquotte, A., et al., A prospective study of
endogenous estrogens and breast cancer in postmenopausal women. J. Natl. Cancer
Inst. 87, 190–197 (1995).