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Chem. Res. Toxicol. 2006, 19, 164-172
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
Muhammad Zahid, Ekta Kohli, Muhammad Saeed, Eleanor Rogan, and Ercole Cavalieri* Eppley Institute for Research in Cancer and Allied Diseases, UniVersity of Nebraska Medical Center, 986805 Nebraska Medical Center, Omaha, Nebraska 68198-6805 Strong evidence supports the idea that specific metabolites of estrogens, mainly catechol estrogen- 3,4-quinones, can react with DNA to become endogenous initiators of breast, prostate, and other humancancers. Oxidation of the catechol estrogen metabolites 4-hydroxyestradiol (4-OHE2) and 2-OHE2 leadsto the quinones, estradiol-3,4-quinone (E2-3,4-Q) and estradiol-2,3-quinone (E2-2,3-Q), respectively. Thereaction of E2-3,4-Q with DNA affords predominantly the depurinating adducts 4-OHE2-1-N3Ade and4-OHE2-1-N7Gua, whereas the reaction of E2-2,3-Q with DNA yields the newly synthesized depurinatingadduct 2-OHE2-6-N3Ade. The N3Ade adducts are lost from DNA by rapid depurination, while the N7Guaadduct is lost from DNA with a half-life of ∼3 h at 37 °C. To compare the relative reactivity of E2-3,4-Qand E2-2,3-Q, the compounds were reacted individually with DNA for 0.5-20 h at 37 °C, as well as inmixtures (3:1, 1:1, 1:3, and 5:95) for 10 h at 37 °C. Depurinating and stable adducts were analyzed. Insimilar experiments, the relative reactivity of 4-OHE2 and 2-OHE2 with DNA was determined afteractivation by lactoperoxidase, tyrosinase, prostaglandin H synthase (PHS), or 3-methylcholanthrene-induced rat liver microsomes. Starting with the quinones, the levels of depurinating adducts formed fromE2-3,4-Q were much higher than that of the depurinating adduct from E2-2,3-Q. Similar results wereobtained with lactoperoxidase or tyrosinase-catalyzed oxidation of 4-OHE2 and 2-OHE2, whereas withactivation by PHS or microsomes, a relatively higher amount of the depurinating adduct from E2-2,3-Qwas detected. These results demonstrate that the E2-3,4-Q is much more reactive with DNA than E2-2,3-Q. The relative reactivities of E2-3,4-Q and E2-2,3-Q to form depurinating adducts correlate with thecarcinogenicity, mutagenicity, and cell-transforming activity of their precursors, the catechol estrogens4-OHE2 and 2-OHE2. This is essential information for understanding the cancer risk posed by oxidationof the two catechol estrogens.
Introduction
Catechol estrogens, 2-hydroxyestrone(estradiol) [2-OHE1(E2)] The initial failure to demonstrate that estrogens induce 1(E2), are among the major metabolites of E1 and mutations in bacterial and mammalian test systems (1-6) 2. If these metabolites are oxidized to the electrophilic catechol estrogen quinones, they may react with DNA. The 4-catechol resulted in the classification of estrone (E1)1 and estradiol (E2) estrogens are carcinogenic in Syrian golden hamsters, as well as epigenetic carcinogens that function mainly by stimulating as CD-1 mice (1, 20, 21), whereas the 2-catechol estrogens are abnormal cell proliferation via estrogen receptor-mediated not carcinogenic in the hamsters (1, 20) and are borderline processes (3, 7-12). The stimulated cell proliferation can result carcinogens in CD-1 mice (21).
in more accumulation of genetic damage leading to carcino- genesis (9, 10, 12).
1(E2) is easily oxidized to catechol estrogen-3,4- Compelling strong evidence has resulted in a new paradigm predominantly depurinating adducts (13-16). These adducts of cancer initiation by estrogens. Discovery that specific generate apurinic sites that may lead to cancer-initiating oxidative metabolites of estrogens can react with DNA (13- mutations (17-19), which transform cells (22-25), thereby 16) led to and supported the hypothesis that estrogen metabolites can become endogenous chemical carcinogens by generatingmutations (17-19) that can lead to initiation of cancer. The To determine the DNA adducts of E1(E2)-3,4-Q, standard initiating mechanism can occur in hormone-dependent and adducts were synthesized by reaction of the quinones with deoxyguanosine (dG), deoxyadenosine (dA), and the nucleobaseAde (16, 26). The reaction of E1(E2)-3,4-Q with dG affordedthe depurinating adduct 4-OHE1(E2)-1-N7guanine (Gua) by 1,4- * To whom correspondence should be addressed. Tel: 402-559-7237.
Fax: 402-559-8068. E-mail: ecavalie@unmc.edu.
Michael addition (26). The reaction of E1(E2)-3,4-Q with dA 1 Abbreviations: Ade, adenine; COSY, chemical shift correlation did not produce any adduct. However, the reaction of these spectroscopy; dA, deoxyadenosine; dG, deoxyguanosine; E1, estrone; E2, quinones with Ade resulted in the formation of 4-OHE1(E2)-1- estradiol; E2-2,3-Q, estradiol-2,3-quinone; E2-3,4-Q, estradiol-3,4-quinone; N3Ade by 1,4-Michael addition (16). The rationale for the FAB-MS, fast atom bombardment tandem mass spectrometry; Gua, guanine;HMBC, heteronuclear multiple bond correlation; HSQC, heteronuclear single formation of N3Ade adducts is described in the Results and quantum coherence; IBX, 2′-iodoxybenzoic acid; LP, lactoperoxidase; MC, 3-methylcholanthrene; NOE, nuclear Overhauser effect; OHE2, hydroxy- When E1(E2)-2,3-Q was reacted with dG or dA, a different estradiol; PDA, photodiode array; PHS, prostaglandin H synthase; TFA,trifluoroacetic acid; TOCSY, total correlation spectroscopy.
profile of adducts from those formed by E1(E2)-3,4-Q was ReactiVity of E2-3,4-Q Vs E2-2,3-Q with DNA Chem. Res. Toxicol., Vol. 19, No. 1, 2006 165
Figure 1. Reaction of E1(E2)-2,3-Q methide with Ade and dA to form the N3Ade and N6Ade adducts and N6dA adducts, respectively.
obtained (26). Reaction of E1(E2)-2,3-Q with dA afforded Materials and Methods
2-OHE1(E2)-6-N6Ade (Figure 1) and with dG yielded 2-OHE1- Caution: 2-OHE2, 4-OHE2, E2-2,3-Q, and E2-3,4-Q are hazard-
(E2)-6-N2dG (26). In these reactions, E1(E2)-2,3-Q did not react ous chemicals and were handled according to NIH guidelines (27).
as quinones, but as their tautomers, the E1(E2)-2,3-Q methide Chemicals, Reagents, and Enzymes. 2′-Iodoxybenzoic acid
(Figure 1). The electrophilic C-6 of the quinone methide reacted (IBX) was synthesized from 2′-iodobenzoic acid as described (28).
with the exocyclic amino group of dA or dG via 1,6-Michael 2-OHE2 and 4-OHE2 were synthesized by reacting E2 with IBX addition to yield the N6dA (Figure 1) and N2dG adducts, which and then separating the mixture of 2-OHE2 and 4-OHE2 by HPLC.
retain the deoxyribose moiety and are referred to as stable 4-OHE2-1-N3Ade and 4-OHE2-1-N7Gua were synthesized bypublished procedures (16, 26). MnO adducts because they remain in DNA unless repaired.
ascorbic acid, ammonium acetate, formic acid, sodium phosphate, In this article, we report the synthesis of the N3Ade adenine (Ade), DMSO-d6, CH3CN (HPLC grade), H2O2, lactoper- depurinating adduct obtained by reaction of E1(E2)-2,3-Q with oxidase (LP, from bovine milk), NADPH, methemoglobin, and Ade. We also report a study of the competitive reaction between tyrosinase (from mushrooms) were purchased from Sigma-Aldrich Chemical Co. (St. Louis, MO). dG and calf thymus DNA were purchased from USB (Cleveland, OH). Prostaglandin H synthase 2,3-Q) with DNA, as well as the metabolic oxidation of mixtures (PHS) and arachidonic acid were purchased from Cayman Chemical of 4-OHE2 (4-hydroxyestradiol) and 2-OHE2 to their quinones (Ann Arbor, MI), and CH3OH was purchased from Merck KGaA by selected enzymes in the presence of DNA to form DNA (Darmstadt, Germany). 3-Methylcholanthrene (MC)-induced rat adducts. These results will be discussed in relation to the liver microsomes were prepared as described earlier (29), containing mechanism of tumor initiation by estrogens.
40 mg protein/mL, with 11.3 nmol cytochrome P450/mg protein.
166 Chem. Res. Toxicol., Vol. 19, No. 1, 2006
Bond Elute Certify II SPE cartridges were purchased from Varian the data were acquired and processed using the CoulArray software package. Peaks were identified by both retention time and peak Activated MnO2 was prepared as previously described (30), by height ratios between the dominant peak and the peaks in the two treating concentrated aqueous KMnO4 with aqueous MnSO4 solu- adjacent channels. The 2-OHE2-6-N3Ade adduct eluted at 14.47 tion kept at 90 °C, until a slight excess of KMnO4 was present, as min, 4-OHE2-1-N3Ade eluted at 16.55 min, and 4-OHE2-1-N7Gua indicated by the pink coloration of the suspension.
eluted at 17.46 min. The depurinating adducts were quantified by Instrumentation. 1. UV. The UV spectra were obtained during
comparison of peak response ratios with known amounts of HPLC by using a photodiode array detector (PDA, Waters 996, Milford, MA) for all synthesized compounds. HPLC separations Synthesis of Standard Adducts. To a stirred solution of 2-OHE2
or 2-OHE1 (0.2 mmol) in 5 mL of CH3CN at -40 °C, activated 2. NMR. NMR spectra were recorded on a Varian Unity-Inova
MnO2 (2 mmol) was added under an argon atmosphere (Figure 1).
500 instrument operating at a resonance frequency of 499.8 MHz After 10 min, the yellowish green solution was filtered through a for 1H and 125.6 MHz for 13C spectra at 25 °C. Samples were 0.45 Gelman acrodisc directly into a flask containing a solution of dissolved in 600 µL of DMSO-d signals at 2.5 ppm for 1H and 39.7 ppm for 13C. All two-dimensional mixture was stirred overnight at room temperature. It was evapo- (2D) experiments were performed by using the standard Varian rated to dryness, and the residue was redissolved in dimethyl- software (VNMR v6.1c). For 2D experiments, relaxation delays formamide/CH3OH (1:1, 3 mL) and then subjected to preparative HPLC for purification of the adducts.
2 were recorded for a spectral width of 8000 Hz in 2-6-N3Ade. Yield 43%. UV:
two dimensions. 1H-1H correlations were recorded using correlation NMR (ppm): 8.93 (s, 1H, OH, exchangeable with D2O), 8.77 (s, spectroscopy (COSY) and total correlation spectroscopy (TOCSY) 1H, OH, exchangeable with D2O), 8.17 (s, 1H, 8-H-Ade), 7.47 (s, experiments. The TOCSY experiment was performed in the states 1H, 2-H-Ade), 7.20 (s, 2H, NH2-Ade, exchangeable with D2O), TPPI mode with a MLEV17 spin lock at 10 kHz field strength. In 6.80 (s, 1H, 1-H), 6.30 (s, 1H, 4-H), 5.65 (d, 1H, J ) 4.0 Hz, pulsed field gradient 1H-13C heteronuclear single quantum coher- 6-H), 4.45 (s, 1H, 17 -OH, exchangeable with D2O), 3.49 (t, 1H, ence (HSQC) and heteronuclear multiple bond correlation (HMBC) J ) 8.5 Hz, 17R-H), 2.20 (dd, 1H, J ) sequences, delays were optimized for coupling constants around 2.13 (m, 1H), 1.96 (d, 1H, J ) 14.0 Hz), 1.75-1.91 (m, 4H), 1.47- 140 and 8 Hz, respectively. One-dimensional nuclear Overhauser 1.52 (m, 2H), 1.38-1.40 (m, 1H), 1.13-1.28 (m, 3H), 0.61 (s, (NOE) experiments were recorded in difference mode by subtracting 3H, CH3). 13C NMR (ppm): 156.0, 152.2, 149.3, 145.8, 144.0, one spectrum with irradiation on resonance from another one 140.1, 132.7, 122.8, 118.8, 116.0, 112.6, 80.0, 51.3, 48.8, 43.3, without irradiation with a relaxation delay of 5 s.
43.1, 36.6, 33.9, 33.5, 29.9, 25.8, 22.7, 11.3. FAB-MS [M + H]+: 3. Mass Spectrometry. Fast atom bombardment tandem mass
422.2170 calcd for C23H28N5O3; observed, 422.2192.
spectrometry (FAB-MS) was conducted at the Nebraska Center for 2. 2-OHE2-6-N6Ade. Yield 9%. UV: λmax 214, 277 nm. 1H NMR
Mass Spectrometry (University of Nebraska-Lincoln) using a (ppm): 9.25 (br.s, 2H, OH, exchangeable with D2O), 8.23 (s, 1H, MicroMass (Manchester, England) AutoSpec high-resolution mag- 8-H-Ade), 8.07 (s, 1H, 2-H-Ade), 7.73 (br.s, 1H, 6-NH-Ade, netic sector mass spectrometer. The instrument was equipped with exchangeable with D2O), 6.67 (s, 1H, 1-H), 6.55 (s, 1H, 4-H), 5.42 an orthogonal acceleration time-of-flight serving as the second mass (br.s, 1H, 6-H), 4.40 (s, 1H, 17 -OH, exchangeable with D2O), spectrometer. Xenon was admitted to the collision cell at a level to 3.53 (t, 1H, J ) 8.5 Hz, 17R-H), 2.09-2.19 (m, 1H), 1.95-2.04 attenuate the precursor ion signal by 75%. Data acquisition and (m, 1H), 1.73-1.93 (m, 4H), 1.29-1.58 (m, 4H), 1.06-1.25 (m, processing were accomplished using OPUS software that was 3H), 0.74 (s, 3H, CH3). FAB-MS [M + H]+: 422.2170 calcd for provided by the manufacturer (Microcasm). Samples were dissolved in 5-10 µL of CH3OH; 1 µL aliquots were placed on the sample 3. 2-OHE1-6-N3Ade. Yield 41%. UV: λmax 214, 264 nm. 1H
probe tip along with 1 µL of a 1:1 mixture of glycerol/thioglycerol.
NMR (ppm): 8.97 (s, 1H, OH, exchangeable with D2O), 8.77 (s, 4. HPLC. Preparative HPLC was conducted on a Luna-2 C-18
1H, OH, exchangeable with D2O), 8.17 (s, 1H, 8-H-Ade), 7.58 (s, column (10 µm, 120 Å, 21.2 mm × 250 mm, Phenomenex, 1H, 2-H-Ade), 7.19 (s, 2H, NH2-Ade, exchangeable with D2O), Torrance, CA) on a Waters 600E solvent delivery system equipped 6.78 (s, 1H, 1-H), 6.27 (s, 1H, 4-H), 5.70 (d, 1H, J ) 4.5 Hz, with a 996 PDA by using a linear gradient of 10% CH 6-H), 4.45 (s, 1H, 17 -OH), 2.20 (dd, 1H, J ) 10.5, J ) 1.5 Hz), [0.4% trifluoroacetic acid (TFA)] for 5 min, followed by a linear 2.11-2.13 (m, 1H), 1.96 (d, 1H, J ) 14.0 Hz), 1.75-1.91 (m, 4H), 1.47-1.52 (m, 2H), 1.38-1.40 (m, 1H), 1.13-1.28 (m, 3H), 3CN over 45 min at a flow rate of 6 mL/min.
Analytical HPLC was conducted on a Waters 2690 (Alliance) 0.61 (s, 3H, CH3). FAB-MS [M + H]+: 420.2025 calcd for Separations Module equipped with a Waters 996 PDA interfaced to a Digital Venturis Fx 5100 computer by using a Luna-2 C-18 4. 2-OHE1-6-N6Ade. Yield 7%. UV: λmax 210, 277 nm. 1H NMR
column (5 µm, 120 Å, 250 mm × 4.6 mm, Phenomenex). A linear (ppm): 9.20 (br.s, 2H, OH, exchangeable with D2O), 8.22 (s, 1H, gradient of 30% CH3CN/70% H2O (0.4% TFA) to 100% CH3CN 8-H-Ade), 8.04 (s, 1H, 2-H-Ade), 7.79 (br.s, 1H, 6-NH-Ade, in 45 min at a flow rate of 1 mL/min was used to separate the exchangeable with D2O), 6.65 (s, 1H, 1-H), 6.54 (s, 1H, 4-H), 5.50 reaction mixtures. The concentrations and purity of quinones were (br.s, 1H, 6-H), 2.34-2.39 (m, 1H), 2.11-2.13 (m, 1H), 1.94- checked by using a linear gradient starting from 30% CH3CN/H2O 2.06 (m, 4H), 1.81-1.87 (m, 1H), 1.74-1.77 (m, 1H), 1.58-1.62 (0.4% acetic acid) to 100% CH3CN at a flow rate of 1 mL/min in (m, 1H), 1.33-1.53 (m, 4H), 0.87 (s, 3H, CH3). FAB-MS [M + 15 min with UV detection at 280 and 432 nm, respectively. The H]+: 420.2025 calcd for C23H26N5O3; observed, 420.2038.
retention time for E2-3,4-Q was 5.75 min and for E2-2,3-Q, 5.32 Preparation of Quinones. 2-OHE2 (2.5 mg, 8.7 µmol) was
min. Analyses of depurinating adducts were conducted on an HPLC dissolved in 500 µL of CH3CN and stirred at -40 °C. Then, system equipped with dual ESA model 580 solvent delivery activated MnO2 (7.5 mg, 86.2 µmol) was slowly added. After 15 modules, an ESA model 540 autosampler, and a 12 channel min, the yellowish green solution was filtered through a Gelman CoulArray electrochemical detector (ESA, Chelmsford, MA). The acrodisc. Because of the instability of E2-2,3-Q, the quinone in CH3- two mobile phases used were as follows: A, CH3CN:CH3OH:0.1 CN was directly used in an experiment as quickly as possible.
M HCOONH4 (pH 3.7), 15:5:80; and B, CH3CN:CH3OH:0.1 M 4-OHE2 (2.5 mg, 8.7 µmol) was dissolved in 500 µL of CH3CN HCOONH4 (pH 3.7), 50:20:30. The linear gradient changed from and stirred at 0 °C. Then, activated MnO2 (7.5 mg, 86.2 µmol) 100% A to 90% B in 50 min. The serial arrays of 12 coulometric was slowly added. After 20 min, the solution was filtered through electrodes were set at potentials of -10, 100, 150, 210, 270, 330, a Gelman acrodisc and an equal amount of DMSO was added. The 390, 450, 510, 570, 630, and 690 mV. The 50 µL injections were CH3CN was evaporated in a high vacuum rotavapor using dry ice carried out on a Phenomenex Luna-2 C-18 column (5 µm, 120 Å, and acetone in the condenser. After evaporation of CH3CN, the 4.6 mm × 250 mm) at 1 mL/min. The system was controlled, and quinone in DMSO was used for the experiments. The final ReactiVity of E2-3,4-Q Vs E2-2,3-Q with DNA Chem. Res. Toxicol., Vol. 19, No. 1, 2006 167
Figure 2. Depurinating adducts formed after reaction of E2-3,4-Q and E2-2,3-Q (1:1) with DNA. After 10 h, the level of stable adducts was <1.0
µmol/mol DNA-P and <0.5% of total adducts. Mixtures containing 0.87 mM E2-3,4-Q + E2-2,3-Q and 3 mM DNA in 0.067 M sodium potassium
phosphate, pH 7.0, were incubated at 37 °C. At the indicated times, DNA was precipitated with 2 volumes of ethanol and the depurinating adducts
in the supernatant were analyzed.
concentration of quinone (either individually or together) in the removed, and the DNA was analyzed for stable DNA adducts (31).
From the remaining incubation mixture, DNA was precipitated with Covalent Binding of E2-2,3-Q or E2-3,4-Q to DNA. Freshly
2 volumes of ethanol, and the supernatant was used for the analysis prepared E2-2,3-Q and E2-3,4-Q (0.87 mM total concentration in of depurinating adducts, as described above. Control reactions were 0.5 mL of DMSO) individually, as well as in mixtures (Figure 2), carried out under identical conditions with either no enzyme or no were mixed with DNA (3 mM in 0.067 M sodium potassium phosphate buffer, pH 7.0) and incubated at 37 °C for different timeperiods (0.5, 1, 2, 3, 5, 10, 15, and 20 h). At the indicated times, Results and Discussion
DNA was precipitated with two volumes of ethanol, and thesupernatant, containing depurinating adducts, was concentrated to Synthesis and Characterization of Standard Depurinating
1 mL under low pressure, extracted by using a vac-Elute system Adducts. The reaction of E2-2,3-Q with Ade yielded the major
with 1 mL of CH3OH/0.1 M NH4OAc (80:20), and finally analyzed depurinating adduct, 2-OHE2-6-N3Ade (43%), and a minor for depurinating adducts by HPLC with an electrochemical detector, product, 2-OHE2-6-N6Ade (9%) (Figure 1). The reaction of E2- as described above. The levels of depurinating adducts were determined by comparing peak heights with known adduct stan- The reaction with dA or Ade does not occur in ring A of the dards. Precipitated DNA from a 1 mL aliquot from each experimentwas separated for 32P-postlabeling analysis of stable adducts after estrogen, because of the weak electrophilicity of positions 1 purification of the DNA (31).
and 4. However, after tautomerization of the E1(E2)-2,3-Q to Covalent Binding of 2-OHE
2 and 4-OHE2 to DNA. Mixtures
1(E2)-2,3-Q methide, the reaction takes place at C-6 via 1,6- containing different relative concentrations of 2-OHE2 and 4-OHE2 Michael addition. The N3Ade adduct could not be obtained by (0.87 mM total concentration, Figures 3-7) were incubated with reaction of E2-2,3-Q with dA because electrophilic attack of DNA in the presence of different enzymes, including tyrosinase, the C-6 of E2-2,3-Q methide at the nucleophilic N-3 group of LP, PHS, and MC-induced rat liver microsomes. The reaction dA is hindered by the presence of the deoxyribose moiety bound volume in each experiment was 10 mL. In the tyrosinase experi- to the adjacent N-9 in dA. The dA also cannot react with E2- ments, the mixture containing 3 mM calf thymus DNA in 0.067 M 3,4-Q (3), hexestrol-3′,4′-quinone (32), and polycyclic aromatic sodium-potassium phosphate (pH 7.0), 0.87 mM 2-OHE2 or hydrocarbons (33-36) to form N3Ade adducts. With DNA, 4-OHE2 (2.5 mg in 500 µL of DMSO), and 1 mg of enzyme (2577units) was incubated at 37 °C for 2 or 10 h. For the LP-catalyzed instead, N3Ade adducts are formed with estrogens (16, 32) and reaction, the mixture containing 3 mM calf thymus DNA, 0.87 mM aromatic hydrocarbons (37, 38) and rapidly lost by depurination, because the configuration of the deoxyribose moiety in DNA 2 or 4-OHE2 (2.5 mg/ in 500 µL of DMSO), H2O2 (0.5 mM), and 1 mg of enzyme (97 units) was incubated at 37 °C for 2 or 10 h. For PHS-catalyzed reactions, the mixture containing 3 mM DNA, The 1H NMR spectrum of 2-OHE2-6-N3Ade showed two 0.87 mM 2-OHE2 or 4-OHE2 (2.5 mg in 500 µL of DMSO), 1 mL singlets at 8.93 and 8.77 ppm for the two hydroxy groups in of methemoglobin (2.95 mg/mL in 75 mM KH2PO4, pH 7.5), 1 the catechol moiety, which were confirmed by D2O exchange.
mL of arachidonic acid (50 mM), and 800 µL of PHS (400 units) The two single proton integration signals at 8.17 and 7.47 ppm was incubated at 37 °C for 2 or 10 h. For the microsome-catalyzed were assigned to H-8 (Ade) and H-2 (Ade), respectively, based reaction, the 10 mL mixture containing 3 mM DNA in 150 mMTris-HCl (pH 7.5), 150 mM KCl, 5 mM MgCl on similar values in other N3Ade adducts (16, 32, 34-36). In fact, the significant upfield shift of the H-2 (Ade) proton at 7.47 2 (2.5 mg in 500 µL of DMSO), 10 mg of microsomal protein (40 mg/mL), and NADPH (0.6 mM) was incubated at 37 ppm, as compared to H-2 in unsubstituted Ade, strongly °C for 2 or 10 h. A 1 mL aliquot from each reaction mixture was indicates that this proton is shielded by the aromatic ring of the 168 Chem. Res. Toxicol., Vol. 19, No. 1, 2006
Figure 3. Depurinating adducts formed by mixtures of E2-3,4-Q and E2-2,3-Q at different ratios after 10 h of reaction with DNA. The level of
stable adducts formed in the mixtures ranged from 0.1 to 1% of total adducts. Mixtures containing 0.87 mM E2-3,4-Q + E2-2,3-Q and 3 mM DNA
in 0.067 M sodium potassium phosphate, pH 7.0, were incubated at 37 °C. At the indicated times, DNA was precipitated with 2 volumes of ethanol
and the depurinating adducts in the supernatant were analyzed.
estrogen moiety. This corroborates substitution at N-3 in the C-17 hydroxy group and one triplet at 3.53 ppm for the C-17 proposed structure. The two signals at 6.80 and 6.30 ppm were proton in the estrogen moiety were also present. The presence assigned as the aromatic protons H-1 and H-4, respectively, of a broad singlet at 7.53 ppm corresponding to one proton based on 1D and 2D NMR studies, which ruled out the suggests that the 6-NH is substituted in the proposed structure.
possibility of the substitution of Ade in the aromatic ring (at The 1H NMR spectra of 2-OHE1-6-N3Ade and 2-OHE1-6-N6- C-1 and C-4) of E2. Furthermore, the spectrum showed the Ade are very similar to those of 2-OHE2-6-N3Ade and 2-OHE2- doublet of one proton at 5.65 ppm, which correlated to a methine 6-N6Ade, except that the former two show no signals for the group at 51.3 ppm in the HSQC experiment. These 1H and 13C C-17 hydroxy group and C-17 proton. Thus, the structure of chemical shifts suggested a substitution of the base at the C-6 2-OHE1-6-N3Ade and 2-OHE1-6-N6Ade is also established. All position of E2. This is in accordance with the earlier observation four compounds showed the desired exact [M + H]+ in their that E1-2,3-Q undergoes tautomerization to its quinone methide (26); thus, the value of 5.65 ppm was assigned to the H-6 of Relative Reactivity of E2-3,4-Q and E2-2,3-Q with DNA.
E2. Furthermore, the resonance of this proton shows COSY Different concentrations of E2-3,4-Q were reacted with 3 mM correlation with aliphatic protons of the steroid skeleton at 1.76 DNA to examine the saturation level in the formation of ppm, assigned as H-7 in COSY and TOCSY experiments. In depurinating adducts (4-OHE2-1-N3Ade and 4-OHE2-1-N7Gua).
addition, H-7 showed correlations in the COSY spectrum with The level of 0.87 mM E2-3,4-Q was found to be saturating for two other protons, which give a mutual multiplet at 1.74-1.87 measurement of the rate of reaction between E ppm. The broad signal at 7.20 ppm, exchangeable with D corresponding to two protons in the 1H NMR spectrum, excludedthe possibility of attachment of the estrogen moiety at the NH Incubations were conducted at 37 °C for 10 h because the of Ade. This was further confirmed by an NOE experiment, in N7Gua adduct depurinates slowly. When E2-3,4-Q was reacted which irradiation of the signal at δ 6.30 (H-4) produced a strong with DNA for 10 h, the adducts 4-OHE2-1-N3Ade and 4-OHE2- effect on the signal at δ 7.47 (H-2, Ade) and vice versa. These 1-N7Gua were detected at approximately equal levels of 130- extensive NMR studies unequivocally establish the structure of 140 µmol/mol DNA-P (data not shown).
the compound as 2-OHE2-6-N3Ade. Although the nucleophilic The slow depurination of 4-OHE2-1-N7Gua is clearly seen attack of the N-3 position of Ade can take place at either face in Figure 2, in which equal amounts of E2-3,4-Q and E2-2,3-Q at C-6 of the estrogen quinone methide, we obtained were reacted with DNA and the depurinating adducts were only one product, in which the attachment occurred at the R analyzed at various time points. The binding of E2-3,4-Q to face (from below the estrogen plane). Attack at the upper face DNA and depurination of the N3Ade adduct was complete appears to be hindered by the methyl group at C-13.
within 1 h, suggesting that depurination was instantaneous. In The 1H NMR spectrum of 2-OHE2-6-N6Ade showed a broad contrast, the depurination of the N7Gua adduct had a half-life singlet integrated as two protons at 9.25 ppm for the two of approximately 3 h at 37 °C and was complete in 10 h. This hydroxy groups, exchangeable with D2O. Three singlets of one slow loss of deoxyribose was previously observed in the reaction proton each at 8.23, 8.07, and 7.73 ppm were assigned to the of E2-3,4-Q with dG to form the N7Gua adduct (39). The adduct H-8, H-2, and NH of the Ade moiety, respectively. Two singlets 2-OHE2-6-N3Ade was formed at a much lower level, 4 µmol/ at 6.67 and 6.55 ppm were assigned to the H-1 and H-4 protons mol DNA-P, and depurination was also immediate (Figure 2).
of the E2 moiety, respectively; one broad singlet at 5.42 ppm The level of stable adducts determined by the 32P-postlabeling (one proton) for the H-6 of the E2 moiety strongly suggests technique was <0.5% of the total adducts formed (data not substitution at the C-6 position; one singlet at 4.40 ppm for the ReactiVity of E2-3,4-Q Vs E2-2,3-Q with DNA Chem. Res. Toxicol., Vol. 19, No. 1, 2006 169
Figure 4. Depurinating adducts formed in the presence of tyrosinase by mixtures of 4-OHE2 and 2-OHE2 at different ratios after 10 h of reaction
with DNA. The levels of stable adducts formed in the mixtures ranged from 0.1 to 0.7% of total adducts. Mixtures containing 0.87 mM 4-OHE2
+ 2-OHE2, 3 mM DNA, and 0.1 mg/mL tyrosinase in 0.067 M sodium potassium phosphate, pH 7.0, were incubated at 37 °C. At the indicatedtimes, DNA was precipitated with 2 volumes of ethanol and the depurinating adducts in the supernatant were analyzed.
Figure 5. Depurinating adducts formed in the presence of LP by mixtures of 4-OHE2 and 2-OHE2 at different ratios after 10 h of reaction with
DNA. The levels of stable adducts formed in the mixtures ranged from 0.2 to 0.8% of total adducts. Mixtures containing 0.87 mM 4-OHE +
2-OHE2, 3 mM DNA, and 0.1 mg/mL LP and 0.5 mM H2O2 in 0.067 M sodium potassium phosphate, pH 7.0, were incubated at 37 °C. At theindicated times, DNA was precipitated with 2 volumes of ethanol and the depurinating adducts in the supernatant were analyzed.
This study was further delineated by determining the level (data not shown). Similar relative amounts of the three depu- of the three depurinating adducts formed at different ratios of rinating adducts were obtained when LP was used to activate E2-3,4-Q and E2-2,3-Q (Figure 3). The overwhelming abundance the catechol estrogens (Figure 5). The amounts of stable adducts of the depurinating N7Gua and N3Ade adducts formed by E2- formed in the mixtures were similar, ranging from 0.2 (4-OHE2) 3,4-Q is observed at all ratios of quinones. The levels of the to 0.8% (2-OHE2) of total adducts (data not shown). With three depurinating adducts were similar only with 5% E2-3,4-Q activation by PHS, relatively lower amounts of adducts were and 95% E2-2,3-Q. The level of stable adducts formed in these formed from the 4-OHE2, as compared to activation by mixtures ranged from 0.1% of total adducts with 100% E2-3,4-Q tyrosinase or LP. The level of adducts from 2-OHE2 remained, to 1% of total adducts with 100% E2-2,3-Q (data not shown).
instead, about the same (Figure 6). In this experiment, with 95% Relative Reactivity of Enzyme-Activated 4-OHE2 and
2-OHE2 and 5% 4-OHE2, the levels of the two depurinating 2-OHE2 with DNA. When mixtures of 2-OHE2 and 4-OHE2
adducts from 4-OHE2 were about half the amount of 2-OHE2- at different ratios were reacted with DNA in the presence of 6-N3Ade. The level of stable adducts was a little higher, and tyrosinase, the depurinating adducts from 4-OHE2 were the most they represented 0.5 (4-OHE2) to 4% (2-OHE2) of total adducts abundant. The levels of the depurinating adducts were similar with 95-100% 2-OHE2 (data not shown).
only with 95% 2-OHE2 and 5% 4-OHE2 present (Figure 4).
The activation of 4-OHE2 was relatively lower when MC- The levels of stable adducts detected in the reaction mixtures induced rat liver microsomes were used for activation (Figure were approximately the same, which ranged from 0.1 (with 7). In fact, 100% 4-OHE2 yielded only 15-20 µmol/mol DNA-P 100% 4-OHE2) to 0.7% (with 100% 2-OHE2) of total adducts of 4-OHE2-1-N7Gua and 4-OHE2-1-N3Ade. The 2-OHE2-6- 170 Chem. Res. Toxicol., Vol. 19, No. 1, 2006
Figure 6. Depurinating adducts formed in the presence of PHS by mixtures of 4-OHE2 and 2-OHE2 at different ratios after 10 h of reaction with
DNA. The levels of stable adducts formed in the mixtures ranged from 0.5 to 4.1% of total adducts. Mixtures containing 0.87 mM 4-OHE +
2-OHE2, 3 mM DNA, 0.3 mg/mL methemoglobin, 5 mM arachidonic acid, and 40 units/mL PHS in 0.067 M sodium potassium phosphate, pH 7.0,were incubated at 37 °C. At the indicated times, DNA was precipitated with 2 volumes of ethanol and the depurinating adducts in the supernatantwere analyzed.
Figure 7. Depurinating adducts formed in the presence of MC-induced rat liver microsomes by mixtures of 4-OHE2 and 2-OHE2 at different ratios
after 10 h of reaction with DNA. The levels of stable adducts formed in the mixtures ranged from 0.3 to 11% of total adducts. Mixtures containing
0.87 mM 4-OHE +
2-OHE2, 3 mM DNA, 150 mM KCl, 5 mM MgCl2, 1 mg/mL microsomal protein, and 0.6 mM NADPH in 150 mM Tris-HCl, pH 7.5, were incubated at 37 °C. At the indicated times, DNA was precipitated with 2 volumes of ethanol and the depurinating adducts in thesupernatant were analyzed.
N3Ade was obtained in relatively larger amounts. In fact, at a the formation of stable adducts does not correlate with the ratio of 75% 2-OHE2 to 25% 4-OHE2, similar amounts of the carcinogenic potency of 2-OHE2 and 4-OHE2 (1, 20, 21).
three depurinating adducts were detected (Figure 7), and with95% 2-OHE2, the 2-OHE2-6-N3Ade was observed to be present Conclusions
in about a 3-fold higher amount than the two adducts formedfrom 4-OHE2. With the microsomes, the amount of stable The reaction of E2-3,4-Q with dG forms the depurinating adducts ranged from 0.12 µmol/mol DNA-P with 100% 4-OHE2 adduct 4-OHE2-1-N7Gua (13, 16), whereas the reaction of E2- to 1.57 µmol/mol DNA-P with 100% 2-OHE2, representing 0.3 3,4-Q or E2-2,3-Q with Ade forms 4-OHE2-1-N3Ade (16) or (with 4-OHE2) to 11.4% (with 2-OHE2) of total adducts (data 2-OHE2-6-N3Ade, respectively. The reaction of E2-3,4-Q with not shown). This 10-fold increase in the amount of stable DNA yields 4-OHE2-1-N3Ade, which is rapidly depurinated, adducts formed from 2-OHE2 as compared to 4-OHE2 is similar and 4-OHE2-1-N7Gua, which is depurinated slowly, with a half- to results previously obtained when E2-2,3-Q or E2-3,4-Q was life of about 3 h (Figure 2). The final amounts of the two adducts reacted with DNA (40). In the experiments reported here, the are very similar (130-140 µmol/mol DNA-P). The reaction of level of stable adducts formed from enzyme-activated 4-OHE2 E2-2,3-Q with DNA affords 2-OHE2-6-N3Ade, which depuri- ranged from about the same as to approximately 10-fold less nates immediately. The maximum amount of this adduct is 12 than the amount of stable adducts formed from 2-OHE2. Thus, ReactiVity of E2-3,4-Q Vs E2-2,3-Q with DNA Chem. Res. Toxicol., Vol. 19, No. 1, 2006 171
When mixtures of E2-3,4-Q and E2-2,3-Q (3:1, 1:1, 1:3, and References
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observed in ACI rat mammary glands within 12 h of intramam- (2004) Metabolism and DNA binding studies of 4-hydroxyestradioland estradiol-3,4-quinone in vitro and in female ACI rat mammary millary treatment of the mammary glands with E2-3,4-Q (18).
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Recently, 4-OHE2 was found to be mutagenic in the mammary (17) Chakravarti, D., Mailander, P., Li, K.-M., Higginbotham, S., Zhang, gland of female Big Blue rats after 20 weeks of treatment with H., Gross, M. L., Cavalieri, E., and Rogan, E. (2001) Evidence that aburst of DNA depurination in SENCAR mouse skin induces error- implanted estrogen. Much higher fractions of A f G mutations prone repair and forms mutations in the H-ras gene. Oncogene 20, were found in the mammary glands of the 4-OHE2-treated rats than controls (J. Guttenplan, private communication). Addition- (18) Chakravarti, D., Mailander, P. C., Higginbotham, S., Cavalieri, E. L., and Rogan, E. G. (2003) The catechol estrogen-3,4-quinone metabolites 2, but not 2-OHE2, was found to be mutagenic in induce mutations in the mammary gland of ACI rats. Proc. Am. Assoc. Big Blue rat 2 embryonic cells, and the mutants contained significantly higher fractions of A f G mutations than those (19) Zhao, Z., Kosinska, W., Khmelnitsky, M., Cavalieri, E. L., Rogan, E.
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