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Cancer Research 67, 812, January 15, 2007. doi: 10.1158/0008-5472.CAN-06-2133
© 2007 American Association for Cancer Research
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Endocrinology

Cytochrome P450 1B1–Mediated Estrogen Metabolism Results in Estrogen-Deoxyribonucleoside Adduct Formation

Alexandra R. Belous1, David L. Hachey2, Sheila Dawling1, Nady Roodi1 and Fritz F. Parl1

Departments of 1 Pathology and 2 Pharmacology, Vanderbilt University Medical Center, Nashville, Tennessee

Requests for reprints: Fritz F. Parl, Department of Pathology, Vanderbilt University Medical Center, TVC 4918, Nashville, TN 37232. Phone: 615-343-9117; Fax: 615-343-9563; E-mail: fritz.parl{at}mcmail.vanderbilt.edu.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
The oxidative metabolism of estrogens has been implicated in the development of breast cancer; yet, relatively little is known about the mechanism by which estrogens cause DNA damage and thereby initiate mammary carcinogenesis. To determine how the metabolism of the parent hormone 17ß-estradiol (E2) leads to the formation of DNA adducts, we used the recombinant, purified phase I enzyme, cytochrome P450 1B1 (CYP1B1), which is expressed in breast tissue, to oxidize E2 in the presence of 2'-deoxyguanosine or 2'-deoxyadenosine. We used both gas and liquid chromatography with tandem mass spectrometry to measure E2, the 2- and 4-catechol estrogens (2-OHE2, 4-OHE2), and the depurinating adducts 4-OHE2-1({alpha},ß)-N7-guanine (4-OHE2-N7-Gua) and 4-OHE2-1({alpha},ß)-N3-adenine (4-OHE2-N3-Ade). CYP1B1 oxidized E2 to the catechol 4-OHE2 and the labile quinone 4-hydroxyestradiol-quinone to produce 4-OHE2-N7-Gua and 4-OHE2-N3-Ade in a time- and concentration-dependent manner. Because the reactive quinones were produced as part of the CYP1B1-mediated oxidation reaction, the adduct formation followed Michaelis-Menten kinetics. Under the conditions of the assay, the 4-OHE2-N7-Gua adduct (Km, 4.6 ± 0.7 µmol/L; kcat, 45 ± 1.6/h) was produced 1.5 times more efficiently than the 4-OHE2-N3-Ade adduct (Km, 4.6 ± 1.0 µmol/L; kcat, 30 ± 1.5/h). The production of adducts was two to three orders of magnitude lower than the 4-OHE2 production. The results present direct proof of CYP1B1-mediated, E2-induced adduct formation and provide the experimental basis for future studies of estrogen carcinogenesis. [Cancer Res 2007;67(2):812–7]


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Estrogens were discovered over 75 years ago, and, shortly thereafter, their carcinogenic activity was shown in animal experiments (1–3). Since the 1960s, abundant epidemiologic evidence has been collected that implicates estrogens as prime risk factor for the development of human breast cancer (4–6). Yet, it is unknown how estrogens cause DNA damage and thereby initiate mammary carcinogenesis. One possible mechanism is based on the unique chemical structure of estrogens. Unlike all other steroid hormones, estrogens such as 17ß-estradiol (E2) have an aromatic A-ring, which yields catechols upon oxidation that may be further oxidized to highly reactive semiquinones and quinones (Fig. 1 ). The labile quinones, e.g., 4-hydroxyestradiol-quinone (E2-3,4-Q), readily attack DNA by Michael addition to form adducts, such as the apurinic 4-hydroxyestradiol-1({alpha},ß)-N7-guanine (4-OHE2-N7-Gua) and 4-hydroxyestradiol-1({alpha},ß)-N3-adenine (4-OHE2-N3-Ade; Fig. 1; refs. 7–9). Thus, estrogen quinones seem to share a common feature of many chemical carcinogens; that is, the ability to covalently modify DNA. Support for the carcinogenic activity of estrogens and their oxidative products, the catechol estrogens, comes from experiments in animal models. Treatment with E2 and the catechol 4- and 2-hydroxyestrogens caused kidney cancer in male Syrian hamsters and endometrial cancer in female CD1 mice (10–12). Intramammillary injection of E2-3,4-Q into female rats, followed by excision of mammary tissue 1 h later, revealed the presence of the 4-OHE2-N7-Gua and 4-OHE2-N3-Ade adducts (8, 13). Recently, these adducts have also been detected in normal and malignant human breast tissues by liquid chromatography/mass spectrometry (LC-MS) analysis (14, 15).


Figure 1
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  		Figure 1. CYP1B1-mediated oxidative estrogen metabolism results in estrogen-deoxyribonucleoside adduct formation. CYP1B1 catalyzes the oxidation of E2 to catechol estrogens 2-OHE2 and 4-OHE2 and further to semiquinones and quinones (only E2-3,4-SQ and E2-3,4-Q are shown). In the presence of 2'-deoxyguanosine and 2'-deoxyadenosine, the quinone E2-3,4-Q forms the depurinating adducts 4-OHE2-N7-Gua and 4-OHE2-N3-Ade, respectively.

 
Although these data support a causal relation between estrogen exposure and carcinogenesis, there has been no unequivocal proof for a direct link between parent hormone E2 and DNA damage. Research from several laboratories, including our own, has defined the enzymatic components in the metabolic pathway of estrogen oxidation (16–19). As shown in Fig. 1, cytochrome P450 1B1 (CYP1B1), the principal phase I enzyme in the breast, catalyzes the oxidation of E2 to the catechols 2- and 4-hydroxyestradiol (2-OHE2, 4-OHE2) and further to semiquinones (E2-2,3-SQ, E2-3,4-SQ) and quinones (E2-2,3-Q, E2-3,4-Q). Here, we did in vitro experiments with recombinant purified CYP1B1 to establish a direct connection between the parent hormone E2 and DNA adduct formation. Incubation of CYP1B1 with E2 and 2'-deoxyguanosine or 2'-deoxyadenosine resulted in the time- and concentration-dependent formation of 4-OHE2-N7-Gua and 4-OHE2-N3-Ade. Such experimental proof of estrogen-induced DNA damage with a clear definition of the enzyme-mediated molecular mechanism represents a major step toward explaining the etiologic basis of estrogens as breast cancer risk factor.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Chemicals. E2 and 4-OHE2 were obtained from Steraloids (Newport, RI). Deuterated E2-2, 4, 16, and 16-d4 (E2-d4) and 4-OHE2-1, 2, 16, 16, and 17-d5 (4-OHE2-d5) were obtained from CDN Isotopes (Pointe-Claire, Quebec, Canada). Deoxyribonucleosides and all other chemicals were purchased from Sigma (St. Louis, MO).

Synthesis of estrogen adducts. Adducts were synthesized by first preparing the estrogen quinone followed by the reaction with 2'-deoxyguanosine or adenine (7, 9, 20). For example, 4-OHE2-N7-Gua was obtained by adding 30 mg (330 µmol) of activated MnO2 to 10 mg (35 µmol) of 4-OHE2 with stirring in 2.5 mL of tetrahydrofuran at 4°C. After 10 min, the suspension was passed through a glass syringe equipped with two filters (2.0 and 0.45 µm) in tandem to remove MnO2. The yellow filtrate was added dropwise to a solution of 30 mg (113 µmol) 2'-deoxyguanosine in 1.5 mL acetic acid/water (50:50, v/v). The solution was stirred at room temperature for 4 h, and aliquots were stored at –20°C until purification. We used 4-OHE2-d5 in a similar manner to prepare 4-OHE2-d4-N7-Gua for use as an internal standard. Note that one deuterium atom is lost during the addition of N7 of guanine to C1 of the estrogen quinone. The substitution of N7 destabilizes the glycosidic bond, which leads to loss of the deoxyribose moiety from the base (9). The resulting 4-OHE2-N7-Gua was purified by high-performance liquid chromatography (HPLC) on a 4.6 x 150 mm Zorbax XDB 5 µm C-8 column with a water-acetonitrile gradient using an Agilent 1090 Series HPLC (Agilent, Palo Alto, CA) by monitoring the peak absorbance at 292 nm. Fractions were combined, evaporated to dryness, and weighed. Stock solutions were prepared from the weighed standards, dissolved in methanol/water (50:50), and stored at –20°C.

Expression and purification of recombinant enzymes. Purified recombinant CYP1B1 and NADPH-P450 reductase were prepared as previously described (18, 21).

Enzymatic formation of estrogen adducts. Recombinant CYP1B1 (200 pmol) and NADPH-P450 reductase (400 pmol) were mixed with 60 µg L-{alpha}-dilauroyl-sn-glycero-3-phosphocholine in 0.4 mL of 100 mmol/L potassium phosphate buffer (pH 7.4) containing 5 mmol/L glucose 6-phosphate, 1 mmol/L ascorbate, and 5 mmol/L MgCl2. E2 and 2'-deoxyguanosine or 2'-deoxyadenosine were added as indicated for individual assays. Reactions were carried out in duplicate in 14-mL Falcon tubes and initiated by adding glucose-6-phosphate dehydrogenase (0.5 units/mL) and NADP+ to a final concentration of 0.5 mmol/L. Reactions proceeded for 60 min with gentle shaking at 37°C and then were terminated by adding 0.6 mL ethyl acetate, by vortexing, and by chilling on ice. Internal standard 25 nmol/L 4-OHE2-d4-N7-Gua was added, and samples were transferred to 1.5 mL Eppendorf tubes containing ~50 mg solid NaCl. Samples were vortexed for 0.5 min and then mixed by rotation for 20 min, followed by centrifugation at 14,000 rpm for 5 min. Then, 0.5 mL of the upper ethyl acetate phase was transferred to a 6-mL Falcon tube, avoiding any contamination from the lower aqueous phase. The ethyl acetate extraction was repeated, and both extracts were combined and evaporated under N2. The residue was dissolved in 0.1 mL methanol/water (50:50) and stored overnight at 4°C.

LC-MS analysis of estrogen adducts. HPLC separations were done in a ThermoFinnigan Surveyor Integrated HPLC System with a 1.0 x 150–mm Jupiter 5 µm, C18 300 Å (Phenomenex, Torrance, CA) using an acetonitrile/water/formic acid (4.5:95:0.5 for solvent A and 95:4.5:0.5 for solvent B) gradient system. The gradient was programmed at 80 µL/min from 10% B for 1 min, 95% 15 min, and 10% 9 min. Tandem MS analyses were done by electrospray ionization using a ThermoFinnigan TSQ Quantum triple quadrupole instrument operated in the selected-reaction monitoring mode. The mass transitions 438 -> 272 at 36 eV (4-OHE2-N7-Gua), 442 -> 273 at 34 eV (4-OHE2-d4-N7-Gua), and 422 -> 257 at 40 eV (4-OHE2-N3-Ade) were used with an ion integration time of 250 ms/ion. Nitrogen was used as the nebulization and auxiliary drying gas; the spray voltage was 4.4 kV; and the capillary temperature was set at 300°C. The collision cell was depressurized to 1.5 mTorr with argon. The adduct concentration in the samples was determined by using calibration curves that covered the range of 0.5 to 500 nmol/L using the stock solutions described above.

Enzymatic formation of catechol estrogens and gas chromatography-MS analysis of E2 and catechol estrogens. A duplicate set of enzymatic reactions was done to measure E2 and the catechol estrogens. All reaction components and conditions were identical to those for the adduct formation except that the reaction was terminated by the addition of 2 mL CH2Cl2; E2-d4 was added as internal standard. Samples were analyzed for E2, 2-OHE2, and 4-OHE2 as previously described (18).

Kinetic analysis. Kinetic variables were determined by nonlinear regression analysis using GraphPad Prism (San Diego, CA).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Characterization of apurinic adducts 4-OHE2-N7-Gua and 4-OHE2-N3-Ade. We used gas chromatography (GC)-MS to quantitate E2, 2-OHE2, and 4-OHE2; and LC-MS to quantitate 4-OHE2-N7-Gua and 4-OHE2-N3-Ade. To accomplish the LC-MS quantitation, we synthesized the 4-OHE2-N7-Gua and 4-OHE2-N3-Ade adducts as well as 4-OHE2-d4-N7-Gua as internal standard, and purified them by HPLC. The mass spectrometric analysis showed parent ions with tandem MS fragmentation patterns identical to those reported (8, 9). Specifically, the 4-OHE2-N7-Gua [M + H]+ ion at m/z 438 yielded daughter ions of m/z 272 and 152 (Fig. 2A and B ). Similarly, the 4-OHE2-N3-Ade [M + H]+ ion at m/z 422 yielded daughter ions of m/z 257 and 136 (Fig. 2C and D).


Figure 2
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  		Figure 2. Mass spectrometric analysis of apurinic adducts 4-OHE2-N7-Gua and 4-OHE2-N3-Ade. A, mass spectrum of peak eluted at 5.8 min under HPLC conditions described in Materials and Methods. The signal with the m/z ratio of 438 represents the parent compound 4-OHE2-N7-Gua. B, tandem mass spectrum of the 4-OHE2-N7-Gua adduct (m/z 438) determined with a collision energy of 34 eV, resulting in major daughter ions of m/z 272 and 152. C, mass spectrum of HPLC peak eluted at 6.0 min. The signal with the m/z ratio 422 represents the parent compound 4-OHE2-N3-Ade. D, tandem mass spectrum of the 4-OHE2-N3-Ade adduct (m/z 422) determined with a collision energy of 40 eV, resulting in major daughter ions of m/z 257 and 136.

 
Time dependence. We incubated 100 µmol/L E2 with CYP1B1 in the presence of 100 µmol/L 2'-deoxyguanosine and determined the disappearance of E2; the appearance of 4-OHE2 and 2-OHE2; and the formation of 4-OHE2-N7-Gua at 3, 10, 30, and 60 min. The parent hormone E2 was rapidly metabolized and its concentration decreased to ~10 µmol/L at 10 min followed by a slow decline to ~5 µmol/L at 60 min (Fig. 3A ). The concentration of catechol estrogens increased in a hyperbolic pattern throughout the 60-min time course (Fig. 3B). The concentration of 4-OHE2 exceeded that of 2-OHE2 at all time points, consistent with the known preference of CYP1B1 for the formation of 4-OHE2 (18, 19). The concentration of 4-OHE2-N7-Gua also increased in a time-dependent manner (Fig. 3C). It increased linearly to ~10 min and then began to level off, reflecting the decreasing rate of production of 4-OHE2 and presumably E2-3,4-Q. At 60 min, the concentration of the adduct was 300 times lower than that of the catechol (i.e., 10 nmol/L 4-OHE2-N7-Gua versus 3 µmol/L 4-OHE2). We also determined the time-dependent formation of 4-OHE2-N3-Ade by incubating 100 µmol/L E2 with CYP1B1 in the presence of 100 µmol/L 2'-deoxyadenosine. The concentration of 4-OHE2-N3-Ade increased in a similar manner as the 4-OHE2-N7-Gua adduct. However, the final concentration of 4-OHE2-N3-Ade was lower (i.e., 6.5 nmol/L at 60 min; Fig. 3D).


Figure 3
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  		Figure 3. Metabolism of E2 and formation of catechol estrogens and adducts as a function of time. Each reaction contained 100 µmol/L E2, 0.5 µmol/L CYP1B1, 1.0 µmol/L NADPH P450 reductase, and 100 µmol/L 2'-deoxyguanosine or 2'-deoxyadenosine. Reactions proceeded for 3, 10, 30, and 60 min at 37°C. The concentrations of E2 (A) and 4-OHE2 and 2-OHE2 (B) were determined by GC-MS. The production of 4-OHE2-N7-Gua (C) and 4-OHE2-N3-Ade (D) was analyzed by LC-MS as described in Materials and Methods. Points, mean of two replicate assays.

 
Variation of deoxyribonucleoside concentration. We varied the concentrations of 2'-deoxyguanosine and 2'-deoxyadenosine to determine if the formation of 4-OHE2-N7-Gua and 4-OHE2-N3-Ade, respectively, was increased in proportion. Reaction time (60 min) and E2 concentration (100 µmol/L) were kept constant. The amount of deoxyribonucleoside present in the reaction did not affect the metabolism of E2 or the concentration of catechol estrogen metabolites that remained at 60 min as indicated by their unchanged levels. Figure 4A and B shows levels in the presence of 2'-deoxyguanosine; 2'-deoxyadenosine yielded similar results (not shown). In contrast, the concentration of 4-OHE2-N7-Gua increased linearly from 0.5 nmol/L at 12.5 µmol/L 2'-deoxyguanosine to 7.4 nmol/L at 100 µmol/L 2'-deoxyguanosine (Fig. 4C). The concentration of 4-OHE2-N3-Ade also increased linearly, but to only 5.2 nmol/L at 100 µmol/L 2'-deoxyadenosine (Fig. 4D).


Figure 4
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  		Figure 4. Effect of increasing concentrations of 2'-deoxyguanosine and 2'-deoxyadenosine on the formation of 4-OHE2-N7-Gua and 4-OHE2-N3-Ade, respectively. Each reaction contained 100 µmol/L E2, 0.5 µmol/L CYP1B1, and 1.0 µmol/L NADPH P450 reductase in the presence of 12.5, 25, 50, and 100 µmol/L 2'-deoxyguanosine (dGua) or 2'-deoxyadenosine (dAde). Reactions proceeded for 60 min at 37°C. The concentrations of E2 (A) and 4-OHE2 and 2-OHE2 (B) were determined by GC-MS. The production of 4-OHE2-N7-Gua (C) and 4-OHE2-N3-Ade (D) was analyzed by LC-MS as described in Materials and Methods. Points, means of two replicate assays.

 
Variation of E2 concentration. We varied the concentration of E2 from 1 to 100 µmol/L to determine the effect on the formation of the two adducts. The production of catechol estrogen metabolites increased in a concentration-dependent manner, displaying Michaelis-Menten kinetics with Km values of 50 ± 3 µmol/L for 4-OHE2 and 94 ± 5 µmol/L for 2-OHE2 (Fig. 5A ). Although adduct formation is not part of an enzyme reaction, their production also followed Michaelis-Menten kinetics (Fig. 5B and C). However, there were two noticeable differences: (a) the adduct concentration was two to three orders of magnitude lower and (b) the apparent Km for the adduct formation was significantly lower than that of the 4-OHE2 production (i.e., Km 4.6 ± 0.7 µmol/L for 4-OHE2-N7-Gua and Km 4.6 ± 1.0 µmol/L for 4-OHE2-N3-Ade). Although the Km values for the adducts were the same, their kcat values differed (i.e., 45 ± 1.6/h for 4-OHE2-N7-Gua and 30 ± 1.5/h for 4-OHE2-N3-Ade). Using the kcat/Km ratio to indicate the efficiency of adduct formation, it appears that 4-OHE2-N7-Gua was produced 1.5 times more efficiently than 4-OHE2-N3-Ade under the conditions of the assay.


Figure 5
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  		Figure 5. Effect of increasing concentrations of E2 on the formation of 4-OHE2 and 2-OHE2 (A), 4-OHE2-N7-Gua (B), and 4-OHE2-N3-Ade (C). Each reaction contained 0.5 µmol/L CYP1B1, 1.0 µmol/L NADPH P450 reductase, and 100 µmol/L 2'-deoxyguanosine or 2'-deoxyadenosine in the presence of 1, 2.5, 5, 10, 25, 50, 75, and 100 µmol/L E2. Reactions proceeded for 60 min at 37°C, and products were analyzed by GC and LC-MS as described in Materials and Methods. Points, mean of two replicate assays.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Carcinogenesis is usually viewed as a stepwise process beginning with genotoxic effects (initiation) followed by enhanced cell proliferation (promotion). The main estrogen, E2, is a substrate for CYP1A1 and CYP1B1 and a ligand for the estrogen receptor. In its dual role of substrate and ligand, E2 has been implicated in the development of breast cancer by simultaneously causing DNA damage via its oxidation products, the 2-OH and 4-OH catechol estrogens, and by stimulating cell proliferation and gene expression via the estrogen receptor. Thus, E2 and its oxidative metabolites are unique carcinogens that affect both tumor initiation and promotion (6, 22, 23).

E2 is oxidized to catechol estrogens by CYP1A1 and CYP1B1. These enzymes further oxidize the catechol estrogens to semiquinones and quinones. Catechol estrogens and their estrogen quinones/semiquinones undergo redox cycling, which results in the production of reactive oxygen species that can cause oxidative DNA damage ranging from oxidation of bases to single-strand breakage (24–27). Furthermore, the estrogen quinones form Michael addition products with deoxyribonucleosides (9, 20, 28, 29). Additional reactive intermediates may be produced by isomerization of catechol estrogen o-quinones to p-quinone methides (30). Thus, estrogen metabolism leads to the formation of both oxidative and estrogen DNA adducts, all of which were shown to have mutagenic potential (31–33).

Here, we show that the CYP1B1-mediated oxidation of E2 resulted in the time- and concentration-dependent formation of the 4-OHE2-N7-Gua and 4-OHE2-N3-Ade adducts. It appears that the stepwise reaction sequence comprises several catalytic equilibria as shown in Fig. 6 for the C4 oxidation and 4-OHE2-N7-Gua. In the first oxidation step, E2 is rapidly metabolized to 4-OHE2 as documented in the time course experiment (Fig. 3). 4-OHE2 is a product of the first oxidation reaction while also being a substrate for the second oxidation reaction to E2-3,4-Q. It is unknown how CYP1B1 can accommodate E2, 4-OHE2, and E2-3,4-Q, and whether these compounds change position within the enzyme structure during the stepwise reaction process. Clearly, a fraction of 4-OHE2 is released from the enzyme and can be measured as a stable product (Fig. 3). Estrogen quinones are highly reactive with short half-lives because of the strained 1,2-diketone functionality inherent in o-quinones. For example, the o-quinones formed from 4-OHE1 and 2-OHE1 were determined to have half-lives of 12 min and 42 s, respectively, at pH 7.4 and at 37°C (34). The labile estrogen quinones readily react with a variety of physiologic compounds, ranging from amino acids, such as lysine and cysteine, to the tripeptide glutathione ({gamma}-glutamyl-L-cysteinylglycine), as well as fatty acids and other lipids, and nucleic acids and DNA (17, 35–38). In these reactions, the quinones form Michael addition products with nucleophilic groups, typically yielding a mixture of several stable conjugation products (39, 40). In view of this reactivity, one can postulate that the substrate recognition site of CYP1B1, which is in contact with E2-3,4-Q or E2-2,3-Q, does not contain lysine or cysteine residues as these amino acids would be attacked by the quinones. The resulting stable estrogen quinone-CYP1B1 protein adduct would prevent release of the quinone and thereby inactivate the enzyme. Indeed, sequence analysis of the six putative recognition sites of CYP1B1 reveals that only one site contains a lysine residue in a marginal position (41).


Figure 6
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  		Figure 6. Diagram of reaction sequences involved in CYP1B1-mediated oxidation of E2 to E2-3,4-Q and formation of 4-OHE2-N7-Gua. The transient semiquinone (millisecond half-life; ref. 49) is omitted for the sake of clarity. See Discussion for a description of the individual reaction steps.

 
Estrogen quinones are too labile to be reliably quantified, and, in an earlier study, we used the production of the stable glutathione-estrogen conjugates as surrogate markers. We showed that the estrogen quinones are released from CYP1B1 to form conjugates with glutathione (17). The conjugation reaction was accelerated by the enzyme glutathione S-transferase P1. Here, we show that E2-3,4-Q, upon its release from CYP1B1, forms the 4-OHE2-N7-Gua and 4-OHE2-N3-Ade adducts. Although the adduction is not catalyzed by an enzyme, the reactive quinone E2-3,4-Q becomes available as product of the CYP1B1-mediated oxidation reaction. Therefore, the formation of the 4-OHE2-N7-Gua and 4-OHE2-N3-Ade does not occur linearly but rather follows Michaelis-Menten kinetics (Fig. 5). The shape of the hyperbolic curve differs between 4-OHE2 and the adducts. The apparent Km for the adducts (4.6 ± 0.7 µmol/L for 4-OHE2-N7-Gua and 4.6 ± 1.0 µmol/L for 4-OHE2-N3-Ade) was lower than that for 4-OHE2 (50 ± 3 µmol/L), reflecting the high reactivity of the quinone E2-3,4-Q.

As shown in Fig. 6, the formation of 4-OHE2-N7-Gua is also the result of several reaction equilibria. The C1 of E2-3,4-Q reacts by Michael addition with the N7 of 2'-deoxyguanosine to form 4-OHE2-1-N7-2'-dGua (9). The substitution of N7 destabilizes the glycosidic bond, which leads to loss of the deoxyribose moiety and release of the modified base from the DNA backbone, a process called depurination. The generation of two conformational isomers, {alpha} and ß, is the result of a rotational barrier about the C1-N7 bond. Thus, in one isomer, the guanine moiety is located primarily on the {alpha} side of the estrogen ring system, 4-OHE2-1{alpha}-N7-Gua, whereas the other isomer has the guanine moiety located on the ß side, 4-OHE2-1ß-N7-Gua. Nuclear magnetic resonance analysis showed that the two isomers were formed in a ratio of ~60% ß and 40% {alpha} (9). Similar stereochemical considerations apply to 4-OHE2-N3-Ade with a ratio of ~55% ß and 45% {alpha} (8). These isomers can be distinguished by nuclear magnetic resonance but cannot be separated analytically because their rotation is not completely hindered.

The mechanism of 4-OHE2-N3-Ade formation differs from that of the 4-OHE2-N7-Gua adduct due to the steric hindrance of the deoxyribose moiety in 2'-deoxyadenosine. It is known that the synthesis of 4-OHE2-N3-Ade requires adenine rather than 2'-deoxyadenosine because the adjacent deoxyribose linked to N9 in 2'-deoxyadenosine impairs the approach of the electrophilic E2-3,4-Q to N3 (8). Nevertheless, E2-3,4-Q is capable of forming the 4-OHE2-N3-Ade adduct upon reaction with DNA because the configuration of the deoxyribose moiety in DNA renders the N3 group available (8, 42). The underlying mechanism in the CYP1B1-mediated reaction with E2 and 2'-deoxyadenosine appears to involve the generation of the hydroxyl radical, ·OH, which can cleave the glycosidic bond and produce Ade from 2'-deoxyadenosine (43). The hydroxyl radical is continually generated by redox cycling during the oxidative estrogen metabolism catalyzed by CYP1B1 and NADPH-P450 reductase (25). In a separate experiment, we compared 2'-deoxyadenosine with an equimolar concentration of adenine (100 µmol/L). We observed a 7-fold higher CYP1B1-mediated, E2-derived production of 4-OHE2-N3-Ade with adenine than with 2'-deoxyadenosine (data not shown). This result corroborates the notion that adenine is the real target of the adduction.

Although other phase I enzymes, such as CYP1A2 and CYP3A4, are involved in hepatic and extrahepatic estrogen oxidation, CYP1A1 and CYP1B1 display the highest levels of expression in breast tissue (44, 45). In turn, CYP1B1 exceeds CYP1A1 in its catalytic efficiency as E2 hydroxylase and differs from CYP1A1 in its principal site of catalysis (18, 19). CYP1B1 has its primary activity at the C4 position of E2, whereas CYP1A1 has its primary activity at the C2 position in preference to 4-hydroxylation. The 4-hydroxylation activity of CYP1B1 has received particular attention because of experimental evidence in animal models that 4-OH catechol estrogens are more carcinogenic than the 2-OH isomers (10–12). For these reasons, we selected CYP1B1 as the enzyme to investigate the connection between parent hormone– and estrogen-induced adducts. Phase II enzymes, such as peroxidase or tyrosinase, are capable of oxidizing catechol estrogens; however, they do not recognize E2 as substrate. Thus, studies investigating the role of phase II enzymes in estrogen-DNA adduct formation started with 4-OHE2 or 2-OHE2 as substrates and required the addition of H2O2 or cumene hydroperoxide as cosubstrates (8, 13). Moreover, these studies did not perform kinetic analyses that provide insight into the underlying enzyme mechanism of oxidative estrogen metabolism and estrogen-induced adduct formation.

There is a fundamental difference between 2,3-quinones (E2-2,3-Q) and 3,4-quinones (E2-3,4-Q). 3,4-Quinone–derived adducts, such as 4-OHE2-N7-Gua and 4-OHE2-N3-Ade, are lost from DNA by cleavage of the glycosidic bond, leaving apurinic sites in the DNA backbone. Unrepaired apurinic sites can lead to miscoding during DNA replication and can result in mutations. For example, when a guanine adduct is lost by depurination, leaving an apurinic site in the DNA, the preferential insertion of adenine in the opposite DNA strand leads to a G -> T transversion at the site of the adduct (46, 47). In contrast, the deoxyribose is not lost when E2-2,3-Q is incubated with 2'-deoxyguanosine or 2'-deoxyadenosine. The 2,3-o-quinones isomerize rapidly into the more electrophilic p-quinone methides, which then undergo Michael addition reactions. The linkage occurs between C6 of the steroid B ring and the exocyclic amino group of either 2'-deoxyguanosine or 2'-deoxyadenosine. The resulting adducts, such as 2-OHE2-6({alpha},ß)-N2-deoxyguanosine and 2-OHE2-6({alpha},ß)-N6-deoxyadenosine, are stable and retain the deoxyribose (9). Thus, the 2,3-quinones preferentially form stable adducts, whereas the 3,4-quinones are more likely to produce depurinating adducts. This was shown experimentally in reactions with calf thymus DNA. E1-2,3-Q and E2-2,3-Q produced more stable adducts than E1-3,4-Q and E2-3,4-Q, but the latter produced two to three orders of magnitude higher levels of depurinating adducts than E2-2,3-Q and E1-2,3-Q (13). Thus, when both stable and depurinating adducts were considered together, the 3,4-quinones produced much higher levels of DNA adducts (48). Animal experiments have also shown that the 3,4-catechols and quinones possess much greater carcinogenic activity than their 2,3-counterparts (10–12). For these reasons, we have focused our attention in this study on the E2 -> 4-OHE2 -> E2-3,4-Q pathway of oxidative estrogen metabolism. However, we now have the means to examine the corresponding kinetics occurring simultaneously in the E2 -> 2-OHE2 -> E2-2,3-Q pathway. In future experiments, we plan to synthesize the 2-OHE2-N2-deoxyguanosine and 2-OHE2-N6-deoxyadenosine adducts and assess quantitatively both stable and depurinating adducts formed by CYP1B1-mediated oxidation of the parent hormone E2. As shown in Fig. 5, we already measured the production of the intermediary 2-OHE2 and, therefore, will be able to directly determine the concentration of E2-2,3-Q–derived adducts by LC-MS.

Our results provide direct proof of many earlier studies in animal and human tissues in which estrogen exposure was indirectly implicated as cause of DNA damage. The recent detection of 4-OHE2-N7-Gua and 4-OHE2-N3-Ade in normal and malignant human breast tissues provides additional support for the importance of the oxidative estrogen metabolism in causing DNA damage (14, 15). Our investigation provides the experimental basis for further direct studies of estrogen-induced carcinogenesis.


   Acknowledgments
 
Grant support: NIH grant 1R01CA/ES83752 (F.F. Parl).

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received 6/12/06. Revised 8/22/06. Accepted 10/23/06.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Butenandt A. Über "Progynon" ein krystallisiertes weibliches Sexualhormon. Naturwissenschaften 1929;17:879.
  2. Doisy EA, Thayer SA, Veler CD. The crystals of the follicular ovarian hormone. Proc Soc Exp Med 1929;27:417–9.
  3. Lacassagne A. Hormonal pathogenesis of adenocarcinoma of the breast. Am J Cancer 1936;27:217–28.
  4. Bernstein L, Ross RK. Endogenous hormones and breast cancer risk. Epidemiol Rev 1993;15:48–65.[Free Full Text]
  5. MacMahon B, Feinleib M. Breast cancer in relation to nursing and menopausal history. J Natl Cancer Inst 1960;24:733–53.[Medline]
  6. Parl FF. Estrogens, estrogen receptor and breast cancer. Amsterdam: IOS Press; 2000.
  7. Akanni A, Abul-Hajj YJ. Estrogen-nucleic acid adducts: dissection of the reaction of 3,4-estrone quinone and its radical anion and radical cation with deoxynucleosides and DNA. Chem Res Toxicol 1999;12:1247–53.[CrossRef][Medline]
  8. Li KM, 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 2004;25:289–97.[Abstract/Free Full Text]
  9. Stack DE, Byun J, Gross ML, Rogan EG, Cavalieri EL. Molecular characteristics of catechol estrogen quinones in reactions with deoxyribonucleosides. Chem Res Toxicol 1996;9:851–9.[CrossRef][Medline]
  10. Li JJ, Li SA. Estrogen carcinogenesis in Syrian hamster tissues: role of metabolism. Fed Proc 1987;46:1858–63.[Medline]
  11. Liehr JG, Fang WF, Sirbasku DA, Ari-Ulubelen A. Carcinogenicity of catechol estrogens in Syrian hamsters. J Steroid Biochem 1986;24:353–6.[CrossRef][Medline]
  12. Newbold RR, Liehr JG. Induction of uterine adenocarcinoma in CD-1 mice by catechol estrogens. Cancer Res 2000;60:235–7.[Abstract/Free Full Text]
  13. Cavalieri EL, Stack DE, Devanesan PD, et al. Molecular origin of cancer: catechol estrogen-3,4-quinones as endogenous tumor initiators. Proc Natl Acad Sci U S A 1997;94:10937–42.[Abstract/Free Full Text]
  14. Embrechts J, Lemiere F, Van Dongen W, et al. Detection of estrogen DNA-adducts in human breast tumor tissue and healthy tissue by combined nano LC-nano ES tandem mass spectrometry. J Am Soc Mass Spectrom 2003;14:482–91.[CrossRef][Medline]
  15. Markushin Y, Zhong W, Cavalieri EL, et al. Spectral characterization of catechol estrogen quinone (CEQ)-derived DNA adducts and their identification in human breast tissue extract. Chem Res Toxicol 2003;16:1107–17.[CrossRef][Medline]
  16. Dawling S, Roodi N, Parl FF. Methoxyestrogens exert feedback inhibition on cytochrome P450 1A1 and 1B1. Cancer Res 2003;63:3127–32.[Abstract/Free Full Text]
  17. Hachey DL, Dawling S, Roodi N, Parl FF. Sequential action of phase I and II enzymes cytochrome P450 1B1 and glutathione S-transferase P1 in mammary estrogen metabolism. Cancer Res 2003;63:8492–9.[Abstract/Free Full Text]
  18. Hanna IH, Dawling S, Roodi N, Guengerich FP, Parl FF. Cytochrome P450 1B1 (CYP1B1) pharmacogenetics: association of polymorphisms with functional differences in estrogen hydroxylation activity. Cancer Res 2000;60:3440–4.[Abstract/Free Full Text]
  19. Hayes CL, Spink DC, Spink BC, Cao JQ, Walker NJ, Sutter TR. 17ß-Estradiol hydroxylation catalyzed by human cytochrome P450 1B1. Proc Natl Acad Sci U S A 1996;93:9776–81.[Abstract/Free Full Text]
  20. Akanni A, Abul-Hajj YJ. Estrogen-nucleic acid adducts: reaction of 3,4-estrone o-quinone radical anion with deoxyribonucleosides. Chem Res Toxicol 1997;10:760–6.[CrossRef][Medline]
  21. Hanna IH, Teiber JF, Kokones KL, Hollenberg PF. Role of the alanine at position 363 of cytochrome P450 2B2 in influencing the NADPH- and hydroperoxide-supported activities. Arch Biochem Biophys 1998;350:324–32.[CrossRef][Medline]
  22. Yager JD, Davidson NE. Estrogen carcinogenesis in breast cancer. N Engl J Med 2006;354:270–82.[Free Full Text]
  23. Yue W, Santen RJ, Wang JP, et al. Genotoxic metabolites of estradiol in breast: potential mechanism of estradiol induced carcinogenesis. J Steroid Biochem Mol Biol 2003;86:477–86.[CrossRef][Medline]
  24. Han X, Liehr JG. Microsome-mediated 8-hydroxylation of guanine bases of DNA by steroid estrogens: correlation of DNA damage by free radicals with metabolic activation to quinones. Carcinogenesis 1995;16:2571–4.[Abstract/Free Full Text]
  25. Liehr JG, Roy D. Free radical generation by redox cycling of estrogens. Free Radic Biol Med 1990;8:415–23.[CrossRef][Medline]
  26. Nutter LM, Wu YY, Ngo EO, Sierra EE, Gutierrez PL, Abul-Hajj YJ. An o-quinone form of estrogen produces free radicals in human breast cancer cells: correlation with DNA damage. Chem Res Toxicol 1994;7:23–8.[CrossRef][Medline]
  27. Roy D, Floyd RA, Liehr JG. Elevated 8-hydroxydeoxyguanosine levels in DNA of diethylstilbestrol-treated Syrian hamsters: covalent DNA damage by free radicals generated by redox cycling of diethylstilbestrol. Cancer Res 1991;51:3882–5.[Abstract/Free Full Text]
  28. Bolton JL, Pisha E, Zhang F, Qiu S. Role of quinoids in estrogen carcinogenesis. Chem Res Toxicol 1998;11:1113–27.[CrossRef][Medline]
  29. Bolton JL, Trush MA, Penning TM, Dryhurst G, Monks TJ. Role of quinones in toxicology. Chem Res Toxicol 2000;13:135–60.[CrossRef][Medline]
  30. Bolton JL, Shen L. p-Quinone methides are the major decomposition products of catechol estrogen o-quinones. Carcinogenesis 1996;17:925–9.[Abstract/Free Full Text]
  31. Kuchino Y, Mori F, Kasai H, et al. Misreading of DNA templates containing 8-hydroxydeoxyguanosine at the modified base and at adjacent residues. Nature 1987;327:77–9.[CrossRef][Medline]
  32. Shibutani S, Takeshita M, Grollman A. Insertion of specific bases during DNA synthesis past the oxidation-damaged base 8-oxodG. Nature 1991;349:431–4.[CrossRef][Medline]
  33. Terashima I, Suzuki N, Shibutani S. Mutagenic properties of estrogen-quinone derived DNA adducts in Simian kidney cells. Biochemistry 2001;40:166–72.[CrossRef][Medline]
  34. Iverson SL, Shen L, Anlar N, Bolton JL. Bioactivation of estrone and its catechol metabolites to quinoid-glutathione conjugates in rat liver microsomes. Chem Res Toxicol 1996;9:492–9.[CrossRef][Medline]
  35. Cao K, Devanesan PD, Ramanathan R, Gross ML, Rogan EG, Cavalieri EL. Covalent binding of catechol estrogens to glutathione catalyzed by horseradish peroxidase, lactoperoxidase, or rat liver microsomes. Chem Res Toxicol 1998;11:917–24.[CrossRef][Medline]
  36. Tabakovic K, Abul-Hajj YJ. Reaction of lysine with estrone 3,4-o-quinone. Chem Res Toxicol 1994;7:696–701.[CrossRef][Medline]
  37. Tabakovic K, Gleason WB, Ojala WH, Abul-Hajj YJ. Oxidative transformation of 2-hydroxyestrone. Stability and reactivity of 2,3-estrone quinone and its relationship to estrogen carcinogenicity. Chem Res Toxicol 1996;9:860–5.[CrossRef][Medline]
  38. Wang MY, Liehr JG. Induction by estrogens of lipid peroxidation and lipid peroxide-derived malonaldehyde-DNA adducts in male Syrian hamsters: role of lipid peroxidation in estrogen-induced kidney carcinogenesis. Carcinogenesis 1995;16:1941–5.[Abstract/Free Full Text]
  39. Abul-Hajj YJ, Cisek PL. Regioselective reaction of thiols with catechol estrogens and estrogen-O-quinones. J Steroid Biochem 1986;25:245–7.[CrossRef][Medline]
  40. Abul-Hajj YJ, Tabakovic K, Gleason WB, Ojala WH. Reactions of 3,4-estrone quinone with mimics of amino acid side chains. Chem Res Toxicol 1996;9:434–8.[CrossRef][Medline]
  41. Lewis DFV, Gillam EMJ, Everett SA, Shimada T. Molecular modelling of human CYP1B1 substrate interactions and investigation of allelic variant effects on metabolism. Chem Biol Interact 2003;145:281–95.[CrossRef][Medline]
  42. Zahid M, Kohli E, Saeed M, Rogan EG, Cavalieri EL. The greater reactivity of estradiol-3,4-quinone vs estradiol-2,3-quinone with DNA in the formation of depurinating adducts: Implications for tumor-initiating activity. Chem Res Toxicol 2006;19:164–72.[CrossRef][Medline]
  43. Burrows CJ, Muller JG. Oxidative nucleobase modifications leading to strand scission. Chem Rev 1998;98:1109–51.[CrossRef][Medline]
  44. Huang Z, Fasco MJ, Figge HL, Keyomarsi K, Kaminsky LS. Expression of cytochromes P450 in human breast tissue and tumors. Drug Metab Dispos 1996;24:899–905.[Abstract]
  45. Shimada T, Hayes CL, Yamazaki H, et al. Activation of chemically diverse procarcinogens by human cytochrome P-450 1B1. Cancer Res 1996;56:2979–84.[Abstract/Free Full Text]
  46. Cavalieri EL, Rogan EG. Role of aromatic hydrocarbons in disclosing how catecholestrogens initiate cancer. Adv Pharmacol 1998;42:837–40.
  47. Loeb LA, Preston BD. Mutagenesis by apurinic/apyrimidinic sites. Annu Rev Genet 1986;20:201–30.[CrossRef][Medline]
  48. Stack DE, Cavalieri EL, Rogan EG. Catecholestrogens procarcinogens: depurinating adducts and tumor initiation. Adv Pharmacol 1998;42:833–6.
  49. Kalyanaraman B, Sealy RC, Sivarajah K. An electron spin resonance study of o-semiquinones formed during the enzymatic and autoxidation of catechol estrogens. J Biol Chem 1984;259:14018–22.[Abstract/Free Full Text]



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