Spectral Characterization of Catechol Estrogen Quinone (CEQ

Chem. Res. Toxicol. 2003, 16, 1107-1117

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Spectral Characterization of Catechol Estrogen Quinone (CEQ)-Derived DNA Adducts and Their Identification in Human Breast Tissue Extract Y. Markushin,† W. Zhong,† E. L. Cavalieri,‡ E. G. Rogan,‡ G. J. Small,† E. S. Yeung,† and R. Jankowiak*,† Ames Laboratory - USDOE and Department of Chemistry, Iowa State University, Ames, Iowa 50011; Eppley Institute for Research in Cancer and Allied Diseases, University of Nebraska Medical Center, Omaha, Nebraska 68198-6805 Received April 29, 2003

Estrogens, including the natural hormones estrone (E1) and estradiol (E2), are thought to be involved in tumor induction. Catechol estrogen quinones (CEQ) derived from 4-hydroxyestrone (4-OHE1) and 4-hydroxyestradiol (4-OHE2) react with DNA and form depurinating N7Gua and N3Ade adducts that might be responsible for tumor initiation (Cavalieri, E. L., et al. (2000) J. Natl. Cancer Inst. Monogr. 27, 75). Current detection limits for the CEQ-derived DNA adducts by high-performance liquid chromatography with multichannel electrochemical detection are in the picomole range. To improve the limit of detection (LOD) for CEQ-derived DNA adducts, spectrophotometric monitoring was investigated. Spectroscopic studies of 4-OHE1-1-N3Ade, 4-OHE1-1-N7Gua, 4-OHE2-1-N3Ade, and 4-OHE2-1-N7Gua adduct standards were performed at 77 and 300 K. Upon laser excitation at 257 nm, the 4-OHE1- and 4-OHE2-derived N7Gua and N3Ade adducts are strongly phosphorescent at T ) 77 K. No phosphorescence was observed at 300 K. Both N3Ade and N7Gua adduct types have weak phosphorescence origin bands near 383 and 385 nm, respectively. The corresponding phosphorescence lifetimes are 1.11 ( 0.05 and 0.37 ( 0.05 s. The LOD, based on phosphorescence measurements, is in the low femtomole range. The concentration LOD is approximately 10-9 M, i.e., similar to that recently obtained for CEQ-derived N-acetylcysteine conjugates (Jankowiak, R., et al. (2003) Chem. Res. Toxicol. 16, 304). The LOD in capillary electrophoresis (CE) with field-amplified sample stacking and absorbance detection is about 3 × 10-8 M. To verify whether CEQ-derived DNA adducts are formed in humans or not, tissue extracts from two breast cancer patients were analyzed by CE interfaced with room temperature absorption and low temperature (laser-excited) phosphorescence spectroscopies. For the first time, formation of CEQ-derived DNA adducts is shown in humans. For example, the level of 4-OHE1-1-N3Ade in the breast tissue extract from a patient with breast carcinoma (8.40 ( 0.05 pmol/g of tissue) is larger by a factor of about 30 than that in the breast tissue sample from a woman without breast cancer (0.25 ( 0.05 pmol/g of tissue). In contrast, similar amounts of 4-OHE2-1-N3Ade were observed in both types of tissue. Although more breast tissue samples from women with and without breast cancer need to be studied, these results suggest that the N3Ade adducts could serve as biomarkers to predict the risk of breast cancer.

Introduction Estrogens have been implicated in the etiology of human breast cancer by various types of evidence (1-5). The natural estrogens, E11 and E2, are metabolized at the 2- or 4-position with formation of catechol estrogens. Major metabolic pathways of E1 and E2 are discussed in detail in refs 4 and 5. Estrogens can be converted to catechol estrogens, which, in turn, are metabolized to CEQs (2, 4, 5), presumed to be the ultimate carcinogenic metabolites that can induce mammary, pituitary, cervical, and uterine tumors in rats, mice, and guinea pigs (1). It has been demonstrated that estrogens can become endogenous carcinogens capable of breast tumor initiation by mutation of critical genes (5, 6). Exposure to estrogens is also a risk factor for other human cancers (3). * To whom correspondence should be [email protected]. † Iowa State University. ‡ University of Nebraska Medical Center.

addressed.

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Catechol estrogens are oxidized to CEQ by peroxidases and cytochrome P-450 (7-10). Liehr and Roy (11) and Nutter et al. (12) have shown that redox cycling of CEQ and the corresponding semiquinones can also generate hydroxyl radicals that cause DNA damage. CEQs derived from the catechol estrogens, 4-OHE1 and 4-OHE2, react 1 Abbreviations: Ade, adenine; CE, capillary electrophoresis; CEQ, catechol estrogen quinone(s); Cys, cysteine; DB[a,l]P, dibenzo[a,l]pyrene; DMSO, dimethyl sulfoxide; E1, estrone; E2, estradiol; E2-3,4Q, estradiol-3,4-quinone; F, fluorescence; FASS, field-amplified sample stacking; Gua, guanine; GSH, glutathione reduced form; 2-OH-CE, 2-hydroxy catechol estrogen; 4-OHE1, 4-hydroxyestrone; 4-OHE2, 4-hydroxyestradiol; 4-OHE1-1-N3Ade, 4-hydroxyestrone-derived N3 adenine adduct; 4-OHE2-1-N3Ade, 4-hydroxyestradiol-derived N3 adenine adduct; 4-OHE1-1-N7Gua, 4-hydroxyestrone-derived N7 guanine adduct; 4-OHE2-1-N7Gua, 4-hydroxyestradiol-derived N7 guanine adduct; 4-OHE1-2-NAcCys, 4-hydroxyestrone-derived N-acetylcysteine conjugate; 4-OHE2-2-NAcCys, 4-hydroxyestradiol-derived N-acetylcysteine conjugate; IP, integrated phosphorescence intensity; IF, integrated fluorescence intensity; MM, molecular mechanics; NAcCys, N-acetylcysteine; PAH, polycyclic aromatic hydrocarbons; QM, quantum mechanics; S1, lowest excited singlet state; T1, lowest excited triplet state.

10.1021/tx0340854 CCC: $25.00 © 2003 American Chemical Society Published on Web 08/08/2003

1108 Chem. Res. Toxicol., Vol. 16, No. 9, 2003

with deoxyribonucleosides and DNA to form depurinating adducts at the N7-position of Gua and the N3-position of Ade (2, 4, 5, 13-15). Importantly, the amount of stable adducts derived from the reaction of E2-3,4-Q with DNA was only 0.02% of the amount of depurinating adducts (2, 4-6). The importance of depurinating DNA adducts derived from various PAHs has been already demonstrated (16, 17). For example, DNA adducts formed by DB[a,l]P are converted into H-ras mutations by DNA damage repair errors, suggesting that the apurinic sites produced by depurinatingDB[a,l]P-DNA adducts are converted into preneoplastic mutations in resting cells (16-18). Mutations in the c-H-ras oncogene of DNA in mouse skin papillomas induced by other PAHs were also analyzed (16, 17). The correlation between the depurinating adducts and the mutations suggests that depurinating adducts may be responsible for the tumor-initiating process (16-18). Thus, it is likely that depurinating CEQ-DNA adducts may play a role in endogenous carcinogenesis (5). CEQ-derived GSH (γ-glutamyl-L-cysteinylglycine) conjugates were also identified in in vitro and in vivo experiments (19-23) and are considered to be potentially useful biomarkers for catechol estrogen-induced DNA damage and risk of breast and other cancers. Conjugation with GSH prevents damage to DNA (5, 23); such conjugation is one of the most important detoxification pathways in biological systems. Natural estrogens possess an unsaturated ring, and phenolic substitution results in UV absorption and very weak fluorescence at room temperature (24, 25). 4-OHE1 and 4-OHE2 are also weakly fluorescent (25). As a result, identification of 4-OHE1- and 4-OHE2-derived DNA adducts has been accomplished following derivatization with a suitable fluorescent marker. For example, selectively excited fluorescence line-narrowing spectroscopy has been used for spectral characterization of fluorescently labeled CEQ-derived N7Gua adducts and their identification in rat mammary tissue (15). However, it has recently been shown that 4-OHE1- and 4-OHE2-derived NAcCys conjugates, although weakly fluorescent at 300 K, are strongly phosphorescent at 77 K; for example, the phosphorescence spectra of CEQderived NAcCys conjugates have a weak origin band at ∼383 nm (26). After the conjugates are cooled from 300 to 77 K, the total luminescence intensity of CEQ-derived NAcCys conjugates is increased by a factor of ∼150, predominantly due to phosphorescence enhancement (26). Theoretical calculations revealed, in agreement with the experimental data, that the lowest singlet (S1) and triplet (T1) states of 4-OHE2-2-NAcCys are of n,π* and π,π* character, respectively, leading to a large intersystem crossing yield and strong phosphorescence. The limit of detection (LOD) for CEQ-derived conjugates, based on phosphorescence measurements, is in the low femtomole range (26). Large bodies of spectroscopic experimental and computational data for purines, pyrimidines, and their derivatives, as well as for various amino acids, have been published (27-32). These data include excited state electronic structures investigated theoretically and fluorescence and/or phosphorescence properties. For example, Ade and Gua are phosphorescent when excited in the far UV range (240-280 nm) at 77 K (32-35). Neither Ade nor Gua emits significant fluorescence in the physiologi-

Markushin et al.

cal range of pH 5-8, but they are very weakly fluorescent at low pH (35). It has recently been shown that typical fluorescence lifetimes of DNA nucleotides and nucleosides in aqueous solutions are on the order of ∼1 ps or less, with fluorescence quantum yields of ∼10-4 (36). Extensive QM calculations of the energies of the electronic transitions and the electronic structures of the nucleic acids and many amino acids in their ground and lower excited singlet and triplet states have also been carried out in many different approximations (27, 29, 3739). For example, the lowest energy singlet and triplet states of neutral Ade are of n,π* and π,π* character, leading to large intersystem crossing yield and strong phosphorescence (27). On the basis of available theoretical calculations, the situation is less clear for neutral Gua, for which the n,π* state is the second lowest singlet (27). Therefore, this work explores the applicability of low temperature spectroscopy for the detection and identification of CEQ-derived DNA adducts. Phosphorescence spectraofthe4-OHE1-1-N3Ade,4-OHE1-1-N7Gua,4-OHE21-N3Ade, and 4-OHE2-1-N7Gua analytes are shown for the first time. Phosphorescence spectra of Ade and Gua are shown for completeness. The above CEQ-DNA adducts are strongly phosphorescent when excited with a UV laser at 257 nm at 77 K, which allows sensitive detection and quantitation at low concentrations. We show that differences in phosphorescence spectra and phosphorescence lifetimes, as well as different pH dependence and different electrophoretic mobilities in CE, allow the identification of CEQ-DNA adducts formed in vivo. Extracts of breast tissue from two women were analyzed by CE interfaced with absorption and luminescence spectroscopies. The results show that CEQ-derived DNA adducts can be identified in human breast tissue extracts.

Materials and Methods Caution: CEQs are hazardous chemicals and should be handled carefully in accordance with NIH guidelines. Chemicals and CEQ-DNA Adduct Standards. 4-OHE1 and 4-OHE2 were synthesized according to Dwivedy et al. (7). The 4-OHE1- and 4-OHE2-derived DNA adduct standards were synthesized as previously described (13, 14). Structural analysis of the above standards was accomplished via NMR (13, 14) and MS (13, 14). Ultrapure grade glycerol was obtained from Spectrum Chemical (Gardena, CA). The purity of standards for CEQ-derived DNA adduct standards, originally separated by HPLC, was verified in our laboratory by CE, which possesses higher separation power than HPLC. CE verified the high purity of the standards; thus, one can be confident that the luminescence spectra correspond to pure analytes. Because they are heat and oxygen sensitive, all CEQ-derived DNA adducts were kept for longer storage at -80 °C under an inert atmosphere (N2 or Ar). Samples were dissolved in methanol:buffer (80:20), with the following buffer content: 0.1 M ammonium acetate and 1 mg/L ascorbic acid in Nanopure water, pH 4.5. Tris[hydroxymethyl]aminomethane was purchased from Fisher Scientific (Fairlawn, NJ). Phosphoric acid and polyoxyethylene 8 cetyl ether (C16E8) were obtained from Sigma-Aldrich (St. Louis, MO). Breast Tissue Extracts/CE. Two breast biopsy specimens (nontumor tissue, ∼1 g wet weight) were obtained from the University of Nebraska Medical Center Hospital (40). Extracts were prepared from the specimens, which were weighed, partially thawed, minced, and ground to a fine powder in liquid nitrogen. Ground tissue was suspended in 2 mL of 0.1 M ammonium acetate, pH 4.4, containing 2 mg/mL ascorbic acid; glucuronidase from Helix pomatia (10 000 units, also containing

Spectral Characterization of CEQ-Derived DNA Adducts 900 units of arylsulfatase) was added, and the tissue was incubated for 16 h at 37 °C. After it was incubated, sufficient methanol was added to give a final concentration of 60 vol %, and the mixtures were extracted with 10 mL of hexane to remove any lipids. The methanol extract was diluted with 0.1 M ammonium acetate, pH 4.4, containing 1 mg/mL ascorbic acid, to an approximate final concentration of 30% methanol, and the methanol/water mixture was applied to a Certify II Sep-Pak (200 mg) cartridge. The cartridge was first eluted with 3 mL of the buffer, followed by elutions with 2 mL each of 20, 40, and 70% methanol in buffer, and fractions were collected. To minimize oxidation of the adducts, ascorbic acid was added to the eluting buffer at a concentration of 1 mg/mL. Collected fractions were concentrated and brought to 1 mL final volume before analysis. The first extract (sample 1) was obtained from a woman with breast carcinoma. The second tissue extract (sample 2) was from a woman who did not have breast cancer. Both samples for CE analysis were prepared in 80:20 MeOH:0.1 M ammonium acetate (pH 4.4, with 1 mg/L ascorbic acid). The analysis of sample 1 was done with a P/ACE MDQ CE system (Beckmancoulter, CA) with a photodiode array (PDA) detector for simultaneous detection of electropherograms and UV absorption spectra of separated analytes. A bare fused-silica capillary (Polymicro Technologies, Phoenix, AZ) with 21 cm effective length and 31.2 cm total length (75 mm i.d. and 360 mm o.d.) was used. The running buffer was 0.5% C16E8 in 0.25 mM Trisphosphate (pH 3.5). Before injection, the solvent in the sample was evaporated by vacuum pump, and then, the sample residue was diluted with the same volume of 75 mM H3PO4 solution. The same extract was also separated with an ISCO (Lincoln, NE) model 3140 Electropherograph System and reanalyzed by low temperature luminescence spectroscopy. The analysis of sample 2 was done using an ISCO model 3140 Electropherograph System. In this case, the total length of the capillary (with the same i.d. and o.d.) was 40 cm, with the effective length of about 20 cm. All other experimental conditions were kept the same as in the P/ACE MDQ system discussed above, except that the detection wavelength was set at 214 nm. The CEQ-derived DNA adduct standards and the breast sample extracts were analyzed without and with FASS conditions (41, 42). FASS was used for analyte preconcentration. In the experiments without stacking, the sample was injected with hydrodynamic flow. To achieve reproducible and accurate stacking results, a water plug was injected into the capillary before the sample (at 0.2 psi for 12 s) followed by the electrokinetic injection of breast extract sample (or adduct standards) at +10 kV for 30 s. The applied electric field for separation was 480 V/cm, and the running temperature was 25 °C. The absorption detection was set at the PDA mode to obtain the electropherograms under different UV wavelengths and the absorption spectra of the separated analytes. After each run, the capillary was rinsed with 0.1 M NaOH for 2 min and running buffer for 5 min. Electropherograms were obtained in the absorbance mode. CE-separated DNA adducts were identified based on the characteristic migration times and corresponding absorption spectra. Various detection wavelengths for the CE electropherograms were utilized (e.g., 214, 260, and 276 nm). For definitive adduct identification, CE was interfaced with low temperature, laser-excited, phosphorescence detection using a specially designed low temperature cryostat (43). Luminescence and Absorption Spectroscopy. Luminescence spectra were obtained using an excitation wavelength of 257 nm of a Lexel 95-SHG-257 CW laser. Emission was dispersed by a model 218 0.3 m monochromator (McPherson, Acton, MA), equipped with a 300 G/mm grating, providing a resolution of ∼1 nm and a spectral window of approximately 200 nm. Spectra were detected with an intensified CCD camera (Princeton Instruments, Trenton, NJ) using gated and nongated modes of detection. A fast shutter, operated by a Uniblitz driver control (model SD-12 2B), was synchronized with the CCD camera (ICCD-1024 MLDG-E1) and used for time-resolved

Chem. Res. Toxicol., Vol. 16, No. 9, 2003 1109

Figure 1. Chemical structures of 4-OHE1- and 4-OHE2-derived N3Ade and N7Gua adducts. phosphorescence measurements. Using this setup time-resolved phosphorescence, spectra (∼10-4-10-8 M analyte concentrations) could be measured in 0.5 s intervals with a gate width of 0.5 s. Phosphorescence lifetimes and phosphorescence excitation spectra (for more concentrated samples; ∼10-4 M) were measured at 77 K using a Cary Eclipse fluorescence spectrophotometer (Varian, Inc., Palo Alto, CA). Experimental conditions were as follows: delay time, 0.1 s; gate time, 5 ms; and number of flashes, 50. For off-line spectroscopic measurements, to ensure good glass formation, glycerol (50 vol %) was added to the samples in buffer just prior to cooling to 77 K in a liquid nitrogen optical cryostat with suprasil optical windows. Samples (ca. 20 µL) were contained in suprasil tubes (2 mm i.d.). Luminescence spectra of 4-OHE1/4-OHE2-derived N3Ade and N7Gua adducts were obtained for 10-5-10-8 M concentrations. All spectra were background corrected. Absorption spectra were measured at room temperature using a UV/vis spectrometer (Perkin-Elmer Lambda 18; Perkin-Elmer Instruments, Wellesley, MA).

Results and Discussion We have shown recently that fluorescence of 4-OHE1 and 4-OHE2 is very weak at both room temperature and 77 K (26). Ade and Gua are very weakly luminescent at room temperature but phosphoresce at 77 K (32, 34). Because neither 4-OHE1 nor 4-OHE2 phosphoresce at room temperature or 77 K (26), we explore below the luminescent properties of the 4-OHE1- and 4-OHE2derived DNA adducts. Luminescence Spectra of CEQ-Derived DNA Adducts. Spectroscopic studies of 4-OHE1-1-N3Ade, 4-OHE11-N7Gua, 4-OHE2-1-N3Ade, and 4-OHE2-1-N7Gua adduct standards were performed at 77 and 300 K upon laser excitation at 257 nm. In the following two subsections, we will demonstrate that the above adducts exhibit strong phosphorescence at T ) 77 K. The schematic structures of these adducts are shown in Figure 1. Spectral characteristics of CEQ-derived Ade and Gua adducts are compared to those of the Ade and Gua bases for completeness. 1. Phosphorescence Spectra of Ade and 4-OHE1and 4-OHE2-1-N3Ade Adducts. Curves a and b in Figure 2A are the 77 K normalized phosphorescence spectra of the Ade and 4-OHE1-1-N3Ade adduct obtained in gated mode (0.5 s delay time) in glycerol/water glass (pH 3.5, λex ) 257 nm). Spectrum a has the origin band at 365 nm and intense vibronic bands at 384, 404, and 425 nm, in agreement with literature data (32). A similar spectrum was obtained at pH 6. The phosphorescence spectrum of 4-OHE1-1-N3Ade (curve b) is less structured and slightly broader than that of Ade, with the unre-


Figure 2. (A) Normalized phosphorescence spectra of Ade (spectrum a) and 4-OHE1-1-N3Ade (spectrum b) in glycerol/ water glass obtained at 77 K with an excitation wavelength of 257.0 nm. Both spectra were obtained in the gated (0.5 s delay time of the observation window) detection mode. The phosphorescence origin bands of Ade and 4-OHE1-1-N3Ade are at 365 and ∼383 nm, respectively. (B) Normalized phosphorescence spectra of Gua (spectrum c) and 4-OHE1-1-N7Gua (spectrum d) obtained at identical conditions as spectra shown in panel A. Concentration, ∼10-5 M; pH 3.5. The dashed and solid arrows in spectrum c label the phosphorescence origin bands of two possible tautomers (see text). The origin band of 4-OHE1-1N7Gua is ∼385 nm.

solved phosphorescence origin band and the center of gravity red-shifted by about 20-25 nm. The position of the origin band, estimated from the fourth derivative of spectrum b, is near 383 nm (see solid arrow); the weakly resolved vibronic bands lie near 405, 425, and 448 nm. An identical spectrum was obtained for 4-OHE2-1-N3Ade (data not shown). Both phosphorescence spectra in Figure 2A show a progression in a mode of ∼1400 cm-1 (a dominant aromatic ring frequency), indicating that the emission originates from a π,π* triplet state. Neither Ade nor 4-OHE1-1-N3Ade revealed measurable phosphorescence at room temperature. We note that spectra a and b in Figure 2A, as well as all spectra presented below, are background corrected. Room temperature fluorescence of Ade, 4-OHE1-1-N3Ade, and 4-OHE2-1-N3Ade (c ∼ 10-4 M) was below the detection limit at our experimental conditions; however, fluorescence of Ade, 4-OHE1-1N3Ade, and 4-OHE2-1-N3Ade was observed at 77 K with the fluorescence origin bands at 297, 300, and 300 nm, respectively. For the sake of brevity, we do not present the room temperature absorption and low temperature fluorescence spectra, but the corresponding band maxima (including those for the phosphorescence spectra) of the above analytes are summarized in Table 1. Figure 3A shows that the IP of 4-OHE1-1-N3Ade (curve b) and 4-OHE1-1-N7Gua (curve a) increases with increasing pH up to a pH of about 8 and decreases slightly at higher pH. Although a stronger pH dependence of IP is observed for the 4-OHE1-1-N7Gua adduct (curve a), both Ade and Gua adducts have similar pKa values of ∼6.5. No spectral changes were observed for N3Ade and N7Gua

Markushin et al.

adducts in the pH range of 3-6; however, irreversible spectral changes (most likely due to oxidation) were observed at pH 11 (spectra not shown). Figure 3B shows that in the pH range studied (3-11) the ratio of IP to the IF, IP/IF, for 4-OHE1-1-N3Ade (curve b) increases up to pH 6 (by a factor of ∼34) and decreases at higher pH (T ) 77 K). The IP of 4-OHE1-1-N3Ade at pH 3.5 is ∼18 times higher than IF, which is consistent with the lowest singlet (S1) and triplet (T1) states being of n,π* and π,π* character, respectively. That is, the S1 f T1 intersystem crossing quantum yield is high, resulting in strong phosphorescence (44). A much weaker pH dependence of IP/IF was observed for Ade with the IP/IF of ∼0.9 (at pH 3.5), in agreement with refs 32-35 (data not shown). The literature values of ground state pKa measured potentiometrically (45) by fluorescence titration (32, 46) are 4.25 and 4.2, respectively. Because the 77 K value of pKa for Ade (as measured by phosphorescence spectra) (32) is 5.3, we conclude that at our experimental conditions (pH 3.5) Ade is in the protonated form. Because the pKa value for 4-OHE1-1-N3Ade is ∼6.5, we conclude that spectra a and b in Figure 2A correspond to the protonated forms of Ade and 4-OHE1-1-N3Ade, respectively. Ade can be protonated at the N1-, N3-, N7-, or N9position, while the 4-OHE1-1-N3Ade adduct can be protonated at the N1-, N7-, or N9-position (37). However, a mixture of tautomers cannot be excluded. It has been shown recently that among different tautomers of Ade, the N9H form would be present dominantly in the ground state in aqueous solutions (37). Although the N9H tautomer could dominate the fluorescence of Ade in aqueous media, the N7H form is more fluorescent and can be produced by energy transfer from the N9H form (37). Moreover, on the basis of theoretical calculations, fluorescence is expected to originate from the π,π* excited singlet state of the N7H tautomer since this transition is the lowest lying transition among all of the singlet excited states belonging to different tautomers of Ade (37). Thus, we conclude that under our experimental conditions, fluorescence and phosphorescence emission originate from the N7H tautomer of Ade and 4-OHE1-1N3Ade. The value of the IP/IF ratio for Ade is ∼1 (pH 3.5) and appears to be in agreement with theoretical calculations in refs 27 and 37, where it was demonstrated that the singlet n,π* transition in both tautomers lies at the same energy range as the π,π* transition, with the former being the second lowest state. The calculated energies of the n,π* transitions for the Ade and Gua bases are in qualitative agreement with the experimental data and undoubtedly testify to the presence of the n,π* transition in their first absorption band (47), although their final relative order is still a matter of debate (27, 37). A significantly larger value of the IP/IF ratio at 77 K for the protonated 4-OHE1-1-N3Ade in comparison with the IP/IF ratio obtained for the protonated Ade (at identical experimental conditions) suggests that the lowest singlet and triplet states in 4-OHE1-1-N3Ade are of n,π* and π,π* character. 2. Phosphorescence Spectra of Gua and 4-OHE1and 4-OHE2-1-N7Gua Adducts. Next, we consider the phosphorescence spectra of Gua and 4-OHE1-1-N7Gua, curves c and d of Figure 2B, respectively. These spectra were also obtained at pH 3.5 with an excitation wavelength of 257 nm in the gated mode (with a 0.5 s delay time of the observation window) and therefore show a

Spectral Characterization of CEQ-Derived DNA Adducts


Table 1. Room and Low Temperature Spectral Properties of 4-OHE1, 4-OHE2, Ade, Gua, and 4-OHE1-1-N3Ade, 4-OHE2-1-N3Ade, 4-OHE1-1-N7Gua, and 4-OHE2-1-N7Gua Adductsa absorbance max ( 1 nm

fluorescence max ( 1 nm 300 K

phosphorescence max ( 1 nm

τphos (s)

77 K

77 K

analyte

300 K

300 K

77 K

4-OHE1 or 4-OHE2 Ade Gua 4-OHE1-1-N3Ade or 4-OHE2-1-N3Ade 4-OHE1-1-N7Gua or 4-OHE2-1-N7Gua

240, 280 261 246, 275 230, 276

320 b b b

312 297 321 300

365, 384, 404, 425 360,c 373,c 395, 422 383,c 405, 425, 448

b 2.10 ( 0.05d 0.06 (90%), 1.36 (10%)e 1.11 ( 0.05

240, 292

b

327

385,b 412, 436, 452

0.37 ( 0.05

a Luminescence spectra were obtained with an excitation wavelength of 257 nm in glycerol/water (1/1) matrix. The matrix contained 10 mM phosphate buffer, pH 3.5. Blank entries indicate luminescence not observed. b Below detection limit. c A weak shoulder. d For Ade, τphos is 3.35 ( 0.1 (pH 1) or 2.2 ( 0.1 s (at pH 7) (34). e For Gua, τphos is 1.6 ( 0.1 (pH 1) and 1.42 s (at pH 7) (34).

Figure 3. (A) Effect of pH on IP in glycerol/water glass at T ) 77 K. (B) Phosphorescence to fluorescence intensity ratio (IP/IF) as a function of pH (T ) 77 K). In both panels, curves a and b are for the 4-OHE1-1-N7Gua and 4-OHE1-1-N3Ade adducts, respectively (c ) 10-4 M).

contribution from pure phosphorescence. Figure 3A (curve a) shows that the pKa value measured for N7Gua adducts by phosphorescence titration (at 77 K) is about 6.5. With reference to the pH dependence of the IP/IF ratio, shown in Figure 3B (curve a), we note that this ratio also increases up to a pH of about 8 and decreases at higher pH. Curve a of Figure 3B shows that the IP of N7Gua adducts is about 17 times higher than the IF at pH 6; thus, the IP/IF ratio for N7Gua adducts is smaller by a factor of 2 in comparison with that for N3Ade (see curve b in Figure 3B). Again, no spectral changes for N7Gua adducts were observed for pH < 7. The IP/IF ratio of ∼0.6 obtained for pure Gua at pH 3.5 (spectra not shown) showed that the fluorescence of Gua is slightly more intense than phosphorescence at 77 K. The position of the phosphorescence origin bands of Gua and 4-OHE1-1-N7Gua is not easily discernible. Moreover, in comparison with Ade, the phosphorescence spectrum of Gua does not reveal a clear vibronic structure. In fact, the shape of spectrum c in Figure 2B suggests that Gua may exist as a mixture of a tautomer and neutral Gua since the ground state pKa values of Gua measured potentiometrically (45, 48) and by fluorescence titration (46) are about 3 and 3.4, respectively. The

estimated pKa of Gua at 77 K is about 4 (32). It was reported that the N9H form of Gua in aqueous solutions is more stable than the N7H form (49). A close inspection of spectrum c in Figure 2B reveals the presence of two weak shoulders near 360 and 373 nm that could correspond to two different phosphorescence origin bands, as indicated by the dashed and solid arrows, respectively. The contribution to spectrum c from the neutral Gua and the N9H tautomer of Gua could account for the much larger spectral width observed in spectrum c of Figure 2B, in comparison with phosphorescence of Ade shown in spectrum a of Figure 2A. The above assignment is also in good agreement with theoretical calculations, which showed that the lowest T1 (π,π*) states of protonated Gua (at the N9 position) and neutral Gua lie at 362.5 and 371.2 nm, respectively (27). Decay time studies of the triplet state in Gua, carried out at 77 K, are consistent with the above analysis since two phosphorescence lifetimes were observed for Gua at pH 3.5 (see Table 1). Apparently, at low pH, Gua does not exist as a single species. A further detailed discussion of the nature of various Ade and Gua tautomers is beyond the scope of this paper. In contrast to Gua, the phosphorescence spectrum of the 4-OHE1-1-N7Gua adduct, shown in spectrum d of Figure 2B, is much narrower. This is not surprising, since, as shown above, the pKa value of 4-OHE1-1-N7Gua is about 6.5. Therefore, the 4-OHE1-1-N7Gua adduct at pH 3.5 exists only as a cation. As a result, the contribution from the neutral form of 4-OHE1-1-N7Gua is eliminated, leading to a much narrower phosphorescence spectrum in agreement with experimental observations. Because the N7H tautomer does not exist in the case of N7Gua adducts, the most likely site for protonation is the N9-position. This is in agreement with theoretical calculations (27), which showed that the protonated N9H form of Gua has the lowest S1 and T1 states with n,π* and π,π* character, respectively, which typically leads to a strong phosphorescence. In contrast, in the case of neutral Gua, the lowest n,π* state is the second singlet (27), which most likely leads to stronger fluorescence from the lowest π,π* singlet state. Because the 77 K phosphorescence of protonated 4-OHE1-1-N7Gua is significantly stronger than the combined phosphorescence of the cation and neutral form of Gua (pH 3.5; c ∼ 10-4 M) by a factor of 30, we suggest that in the case of protonated N7Gua adducts the lowest S1 and T1 states are of n,π* and π,π* character. The absorption, fluorescence, and phosphorescence band maxima of Gua and 4-OHE1/4-OHE2derived N7Gua adducts are also summarized in Table 1.


Markushin et al.

Figure 5. Calibration curve showing the IP measured at 77 K in glycerol/H2O glass (10 mM phosphate buffer, pH 3.3, λex ) 257.0 nm) plotted vs concentration (in mol/L) for the 4-OHE11-N3Ade (black circles) and 4-OHE1-1-N7Gua (white circles).

Figure 4. Panels A and B show normalized phosphorescence decay curves obtained at 77 K in glycerol/buffer glass for 4-OHE1-1-N3Ade and 4-OHE1-1-N7Gua adducts, respectively. Curves a and b in both frames were obtained in the absence and in the presence of 0.75 M KI (λex ) 240 nm; λobs ) 415 nm; excitation and emission slit width, 10 nm; c ∼ 10-4 M). τphos values of 4-OHE1-1-N3Ade/4-OHE1-1-N7Gua adducts in the absence and presence of KI are 1.11/0.0014 and 0.37/0.27 s, respectively (see text for discussion). The inset shows an exponential fit (dashed line) to curve b in panel B plotted on a shorter time scale.

Phosphorescence Lifetimes of CEQ-Derived DNA Adducts at 77 K. Figure 4 shows normalized 77 K phosphorescence decay curves obtained for 4-OHE1-1N3Ade and 4-OHE1-1-N7Gua, panels A and B, respectively. Curves a and b in panel A (and B) were acquired in the absence and presence of the external heavy atoms (0.75 M potassium iodide (KI)), respectively. Phosphorescence lifetimes (τphos) were obtained from an exponential fit to the experimental data; for simplicity, only the fit to curve b in Figure 4B (solid line) is shown in the inset. The τphos values of 4-OHE1-1-N3Ade and 4-OHE11-N7Gua, based on integrated time-resolved 77 K spectra, are 1.11 ( 0.05 and 0.37 ( 0.05 s, respectively. τphos values of 4-OHE1-1-N3Ade and 4-OHE1-1-N7Gua decrease in the presence of KI and are 0.0014 ( 0.0005 (1.4 ms) and 0.27 ( 0.05 s, respectively. That is, because of the heavy atom effect, τphos values of N3Ade and N7Gua adducts decreased by a factor of 2130 and 1.4, while their IP (at our experimental conditions) increased by a factor of ∼500 and ∼12, respectively. At this point, two remarks are pertinent as follows: (i) the τphos for both adduct types is longer than that expected for n,π* triplet states (i.e., 0.01-0.1 s (50-54) and (ii) the τphos of N3Ade and N7Gua adducts decreases in the presence of heavy atoms. We note that the phosphorescence lifetime of a n,π* state is very weakly affected by the external heavy atom effect (55). Thus, these observations also support our earlier assignment that the lowest triplet state of both N3Ade and N7Gua adducts is π,π* in character. Differences

observed in fluorescence/phosphorescence characteristics of N3Ade and N7Gua adducts, e.g., a much larger IP/IF ratio or a much larger heavy atom effect on τphos observed for N3Ade in comparison with N7Gua, are not wellunderstood at the present time but could be attributed to different vibronic interactions between the close-lying n,π* and π,π* states, which may lead to significantly different nonradiative decay rates of the lowest excited state (56). Detection Limits. 1. Phosphorescence-Based Detection. A calibration curve (correlation coefficient r2 ) 0.97) obtained for 4-OHE1-1-N3Ade (black circles) and 4-OHE1-1-N7Gua (white circles) in Gly/H2O glass (10 mM phosphate buffer; pH 3.5) is shown in Figure 5, where integrated 77 K phosphorescence intensity is plotted vs concentration (10-5-10-8) in the unit of molarity, M. The concentration LOD for 4-OHE1-1-N3Ade and 4-OHE1-1N7Gua based on phosphorescence measurements is about 10-9 M (defined by a signal-to-noise ratio of 3). Because the laser-excited volume in our experiments was approximately 1 µL, the LOD for the above-discussed DNA adducts is in the low femtomole range. Similar LOD values were obtained for 4-OHE2-1-N3Ade and 4-OHE21-N7Gua (data not shown). Preliminary CE experiments, using CE interfaced with low temperature phosphorescence detection, have shown that the concentration LOD is also about 10-9 M, but the calibration curve over a large concentration range has not been obtained as yet. We note that the LOD based on the off-line measurement of phosphorescence intensity for CEQ-derived DNA adducts is very similar to that established recently for 4-OHE1-2-NAcCys and 4-OHE2-2-NAcCys conjugates (26) and is about 2 orders of magnitude lower than the LOD obtained by HPLC with multichannel electrochemical detection (22). 2. CE with FASS and Absorbance Detection. Figure 6 shows that the CE concentration LOD with online UV absorbance detection for both 4-OHE1-1-N3Ade (diamonds) and 4-OHE1-1-N7Gua (triangles) is about 10-6 M. To improve the LOD, FASS was used, in which the electric field difference between the sample region and the running buffer focuses the analytes of interest at the boundary that separates these two regions (41, 42). Under our experimental conditions, DNA adducts are positively charged; therefore, in the presence of an electric field, adducts are focused into a narrow zone at the front boundary. The water plug provides an empty region to concentrate cations deeper into the capillary


Figure 6. Calibration curve showing the integrated peak area from the CE electropherograms based on absorbance detection at room temperature. Curves a and b correspond to the 4-OHE11-N3Ade (diamonds) and 4-OHE1-1-N7Gua (triangles) adducts, respectively. The inset shows two CE electropherograms for the 4-OHE1-1-N3Ade adduct standard obtained with a concentration (c) equal to 2.4 × 10-6 M (normal hydrodynamic injection; top curve) and c ) 2.4 × 10-8 M (using FASS; lower curve), respectively (see text).

and away from the inlet end. This approach also improved the electropherogram reproducibility. CE chromatograms shown in the inset of Figure 6 correspond to the 4-OHE1-1-N3Ade standard with a concentration of 2.4 × 10-6 (no FASS; top curve) and 2.4 × 10-8 M (with FASS; lower curve), respectively. A comparison of adduct concentrations and the integrated peak areas in the above two curves reveals that the enhancement factor due to stacking is ∼40. Thus, we conclude that the detection limit in CE with FASS approach (absorbance detection) is about 3 × 10-8 M, i.e., ∼30 times lower than that with CE and on-line phosphorescence detection. CE Analysis of Extracts from Human Breast Tissue. Breast tissue extracts from two women, one diagnosed with a breast carcinoma (sample 1) and the other having only normal breast tissue (sample 2), were analyzed by CE with room temperature absorbance and low temperature phosphorescence detection. The identity of the adducts was confirmed by spiking the extract with DNA adduct standards and by absorption and phosphorescence spectra. The best separation conditions for the CEQ-derived DNA adducts present in breast tissue extracts were obtained with FASS and a neutral surfactant (C16E8) as the buffer additive. In this approach, the DNA adducts are separated based upon their different charges and/or different retention factors in the wormlike or rodlike micelles, which act as a dynamic polymer solution (57). The CE separation of a standard mixture of the CEQ-derived DNA adducts (data not shown) revealed that under our experimental conditions (pH 3.5) the adducts of interest migrated in the following order: (i) 4-OHE1-1-N3Ade, (ii) 4-OHE2-1-N3Ade, (iii) 4-OHE11-N7Gua, and (iv) 4-OHE2-1-N7Gua. The same order was observed at pH 2.5; however, at pH 5.0, the migration order was reversed, with 4-OHE1-1-N7Gua and 4-OHE21-N7Gua migrating faster than the corresponding Ade adducts. The latter is most likely due to the fact that N7Gua adducts are more deprotonated (32). The room temperature absorbance-based electropherograms obtained with FASS for sample 1 (detected at 214 nm) are shown in Figure 7. Curves a and b were obtained without and with spiking the sample with the 4-OHE11-N3Ade adduct standard (40 pmol). These data, based


Figure 7. Spectrum a corresponds to the room temperature absorbance-based electropherogram acquired during CE separation of the extract from human breast tissue (sample 1) with FASS. The absorbance intensity was measured at 214 nm. Spectrum b was obtained for the same sample spiked with the 4-OHE1-1-N3Ade adduct standard. The double arrow marks the location of the 4-OHE1-1-N3Ade. Buffer, pH 3.3; 25 mM H3PO4 plus tris-HCl; CE conditions: 30 cm capillary, V ) 20 kV. Injection: the water plug was injected at 0.2 psi for 0.2 min, and subsequently, the sample was injected at 10 kV for 30 s. The inset shows two absorption spectra (c and d) measured for peak 8 in electropherograms a and b, respectively. The peak migration time is ∼8.7 min.

Figure 8. Spectra a and b correspond to the room temperature absorbance-based electropherogram (observation wavelength at 276 nm) acquired during CE separation of the extract from human breast tissue (sample 1) with FASS (curve a) and after spiking with the 4-OHE1-1-N3Ade adduct standard (curve b). The solid arrow marks the location of the 4-OHE1-1-N3Ade. The integrated area of peak 8 (curve b) corresponds to 40 pmol of 4-OHE1-1-N3Ade adduct (see text for details).

on migration times and standard addition, indicate that peak 8 in curve a of Figure 7 corresponds to 4-OHE1-1N3Ade, and its level in sample 1 is about 8.40 ( 0.05 pmol/g of tissue. To further confirm the identity of this peak, the inset shows two on-line measured absorption spectra obtained for peak 8 in curves a and b in Figure 7. Spectrum c, obtained for peak 8 in curve a, with the absorption maximum at 276 nm, is identical to the absorption spectrum d obtained for peak 8 in the spiked electropherogram b. Spectra c and d are also indistinguishable from the off-line measured absorption spectrum of the 4-OHE1-1-N3Ade reference standard in CE buffer (data not shown). To aid in the assignment of peak 8, Figure 8 shows two additional electropherograms obtained at 276 nm. This observation wavelength corresponds to the absorption maximum of the 4-OHE1-1-


N3Ade adduct. Thus, on the basis of the shape of the absorption spectrum (not shown), the relative intensity of peak 8 should increase at this observation window, as observed in curve a of Figure 8. As before, peak 8 in curve a has identical migration time as that obtained for the 4-OHE1-1-N3Ade standard after spiking the tissue extract with the 4-OHE1-1-N3Ade adduct standard (see curve b, Figure 8). The very weak band labeled by a dashed arrow in Figures 7 and 8 originates from a minor solvent contamination (data not shown). Thus, spectra shown in Figures 7 and 8 suggest that peak 8 most likely corresponds to the 4-OHE1-1-N3Ade adduct. We note that curves a and b in Figures 7 and 8 were offset for clarity. Next, we briefly consider the complexity of the electropherograms in Figures 7 and 8. First, many peaks overlap and are not background resolved. Second, their migration time and the relative peak intensity depend on the observation wavelength. For example, peak 1 is better resolved at the 276 nm observation window (Figure 8), while the intensity of peaks 2-4 (with absorption maxima at 280 nm) decreases in the electropherogram obtained at 260 nm (data not shown). Furthermore, peaks 2-4 are not well-resolved at 276 nm; instead, at this wavelength, three other peaks with slightly different migration times appear between peaks labeled 1 and 5 (see Figure 8). The shoulder in electropherogram a of Figure 7, labeled as band 5, is resolved at the 276 nm electropherogram (see curve a of Figure 8) and was significantly more intense at the 260 nm detection window. This peak (with the absorption maximum at 260 nm) most likely originates from one of the DNA depurinated bases. Such analysis illustrates the advantage of wavelength dispersive detection as compared to fixed wavelength detection for qualitative identification of analytes in complex mixtures. Note that the 5-10 min region of the electropherogram (a) in Figure 8 where the peaks from the N3Ade and N7Gua adducts are expected is less complex than the electropherogram (a) in Figure 7. Nevertheless, as a final test to prove that peak 8 corresponds to the 4-OHE1-1-N3Ade adduct, sample 1 was also analyzed by on-line CE interfaced with low temperature (77 K) phosphorescence detection. The 4-OHE1-1-N3Ade adduct has a characteristic phosphorescence spectrum, as discussed in Luminescence Spectra of CEQ-Derived DNA Adducts. The results are shown in Figure 9; electropherograms a and b were acquired as the capillary was translated through the laser excitation region (λex ) 257.0 nm), and resulting luminescence was measured. Curve a was obtained by monitoring total luminescence, whereas curve b was obtained by luminescence detection at 436 ( 3 nm. The observation wavelength at 436 nm is close to the phosphorescence maximum of the 4-OHE1-1-N3Ade standard. A comparison of spectra a and b clearly shows that curve b, obtained at 436 nm, provides a simpler electropherogram. Both curves reveal a well-resolved peak at the capillary position where peak 8 was registered by the absorbance detection. This was accomplished by marking the position of peak 8 on the capillary and placing it quickly in a specially designed low temperature cryostat. This peak is labeled by an asterisk; diffusion of separated analytes appears to be negligible, as witnessed by the ∼2 mm bandwidth along the capillary position. This is in agreement with our previous studies, where it was shown that the average size of the CE-separated plug is ∼3 mm (58). The 77 K phosphorescence spectrum of the asterisk

Markushin et al.

Figure 9. Spectra a and b correspond to the phosphorescencebased electropherograms acquired after during CE separation of the extract from human breast tissue with FASS, measured at 77 K. Curves a and b were obtained with an excitation wavelength of 257.0 nm. The luminescence intensity (in CW mode) of the electropherogram was obtained by integration of the spectral region from 350 to 500 nm (curve a) and at a narrow observation window at 436 ( 3 nm. Spectra c and d in the inset are obtained for the band labeled by an asterisk and the 4-OHE11-N3Ade adduct standard, respectively. The solid arrow marks the phosphorescence origin band. The dashed arrow points to the band that most likely corresponds to the 4-OHE2-1-N3Ade adduct.

Figure 10. CE absorbance-based electropherogram obtained for sample 2 at 214 nm (T ) 300 K). The peaks labeled as 1, 2, and 3 correspond to the 4-OHE1-1-N3Ade, 4-OHE2-1-N3Ade, and 4-OHE2-1-N7Gua adducts, respectively. The solid arrow marks the expected position for the 4-OHE1-1-N7Gua (see text).

labeled peak (spectrum c) is shown in the inset of Figure 9 and is very similar to the phosphorescence spectrum of the 4-OHE1-1-N3Ade standard shown in the same inset as spectrum d. Both spectra were obtained with 0.5 s delay of the observation window; the solid arrow in the inset indicates the position of the weak phosphorescence origin band near 383 nm. Because the spectra shown in the inset of Figure 9 are indistinguishable, we conclude that the peak labeled by an asterisk corresponds to the 4-OHE1-1-N3Ade adduct. Thus, the 4-OHE1-1-N3Ade adduct is formed in breast tissue from a woman with breast carcinoma. The quantitation procedure described above revealed that the level of 4-OHE1-1-N3Ade in sample 1 is about 8.40 ( 0.05 pmol/g of tissue. It is worth pointing out that the peak near 11 mm along the capillary position in Figure 9, based on the migration time and corresponding phosphoresecence spectrum of the peak, is the 4-OHE2-1N3Ade adduct formed at the level of about 0.90 ( 0.05 pmol/g of tissue. No CEQ-derived N7Gua adducts were observed in sample 1. Finally, we note that spectra c and d in Figure 9 have a slightly different intensity distribution of the vibronic bands in comparison with spectrum d of Figure 2, because of a different matrix composition.



Table 2. Analysis of CEQ-Derived DNA Adducts and Conjugates in Human Breast Tissue from Women with and without Breast Cancer (Concentrations in pmol/g of Tissue) sample

4-OHE1-1-N7Gua ((0.05)

4-OHE1-1-N3Ade ((0.05)

4-OHE2-1-N7Gua ((0.05)

4-OHE2-1-N3Ade ((0.05)

4-OHE1-2-NAcCys ((0.05)

1 2

a a

8.40 0.25

a 0.11

0.90 1.18

b 0.27

a

Not observed under our experimental conditions. b Not measured.

Two CEQ-derived N3Ade and one N7Gua adducts were observed in sample 2; we recall that in this case the tissue extract was obtained from a woman with normal breast tissue. Figure 10 shows a typical absorbance-based electropherogram obtained without stacking at 214 nm (T ) 300K). In this sample, a much weaker background was observed, leading to a better resolution of CEseparated molecules. Standard spiking procedures (resulting electropherograms not shown for simplicity) established that the peaks labeled as 1, 2, and 3 correspond to the 4-OHE1-1-N3Ade, 4-OHE2-1-N3Ade, and 4-OHE2-1-N7Gua adducts, respectively. The level of the above adducts is estimated as 0.25, 1.18, and 0.11 pmol/g of tissue, respectively. The solid arrow in Figure 10 indicates the expected position for the 4-OHE1-1-N7Gua adduct. The absence of any peak in this region clearly indicates that 4-OHE1-1-N7Gua is not present in sample 2. Interestingly, the latter adduct was also absent in sample 1. However, it remains to be established whether the differences observed in adducts formed and levels of their concentration in samples 1 and 2 are statistically significant (research in progress). Finally, we note that the 4-OHE1-2-NAcCys conjugate was also observed in the CE electropherogram obtained for sample 2, as confirmed by spiking with the conjugate standard. The quantitation procedure revealed that sample 2 contained 0.27 ( 0.05 pmol of 4-OHE1-2NAcCys/g of tissue. The peak corresponding to the latter analyte is not resolved in the electropherogram shown in Figure 10; to resolve it, different experimental conditions were used (pH 5, 12 mM NaOAc-HOAc, 4% SDS, and 0.5% C16E8; spectra not shown). This conjugate is a decomposition product of 4-OHE1-2-GSH and may be present in the urine of breast or prostate cancer patients at much higher concentrations. Recently, Rogan et al. (40) showed that in 28 breast carcinoma cases analyzed by HPLC with electrochemical detection, the level of all possible CEQ-derived conjugates (4-OHE1-2-NAcCys, 4-OHE2-2-NAcCys, 4-OHE1-2-Cys, 4-OHE1-2-Cys, and 2-OHE1(E2)-(1 + 4)Cys) was about 8.2 ( 7.0 pmol/g of tissue and three times higher than that in control samples. In this work, we did not attempt to identify CEQ-derived conjugates in sample 1. Nevertheless, the data taken together indicate that CE interfaced with online room temperature absorption and low temperature phosphorescence spectroscopies can be used for detection of both CEQ-derived DNA adducts and CEQ-derived conjugates. This suggests the possibility of developing biomarkers of susceptibility to the initiation of breast cancer and strategies to prevent this disease. The relative adduct concentrations (in pmol/g of tissue), obtained for samples 1 and 2, are summarized in Table 2.

Concluding Remarks A spectroscopic study of CEQ-derived adduct standards was performed at 300 and 77 K upon high energy laser excitation at 257 nm. 4-OHE1- and 4-OHE2-derived

N7Gua and N3Ade adducts are strongly phosphorescent at T ) 77 K; no phosphorescence is observed at 300 K. The phosphorescence spectra of the 4-OHE1/4-OHE2derived N3Ade and N7Gua adducts have a weak origin band near 383 and 385 nm, respectively. Furthermore, the phosphorescence intensity of N3Ade and N7Gua adducts (pH 6) is stronger than their fluorescence by a factor of 34 and 17, respectively. Thus, the bulk of the signal at low temperatures, for both adduct types, originates from phosphorescence. Phosphorescence lifetimes of 4-OHE1-1-N3Ade/4-OHE2-1-N3Ade and 4-OHE11-N7Gua/4-OHE2-1-N7Gua are 1.11 and 0.37 s, respectively. These long lifetimes and their shortening in the presence of heavy atoms, as well as the observed frequencies of the phosphorescence vibronic bands, strongly indicate that the lowest triplet state in both adduct types is π,π*. The LOD for CEQ-derived DNA adducts, based on phosphorescence measurements, is in the low femtomole range. The concentration LOD is approximately 10-9 M, comparable to that recently obtained for 4-OHE1derived NAcCys conjugates (26). The LOD in CE with FASS and absorbance detection is about 3 × 10-8 M. Finally, it has been shown for the first time that CEQderived DNA adducts are formed in humans. Breast tissue extracts from two women, one with breast carcinoma and one without, were analyzed by CE with FASS and absorbance detection and CE interfaced with low temperature, laser-excited, phosphorescence detection. The combination of these methodologies provided evidence that depurinating CEQ-derived N3Ade and N7Gua adducts can be identified in breast tissue extracts; the latter adduct type (4-OHE2-1-N7Gua, c ) 0.11 ( 0.05 pmol/g of tissue) was observed only in the tissue sample from a woman with apparently normal breast tissue. The level of the 4-OHE1-1-N3Ade in the breast tissue extract from the woman with breast carcinoma (8.40 ( 0.05 pmol/g of tissue) is larger by a factor of about 30 than that in the tissue sample from a woman without breast cancer (0.25 ( 0.05 pmol/g of tissue). In contrast, similar amounts of the 4-OHE2-1-N3Ade (∼1 pmol/g of tissue) were formed in both types of tissue. These findings provide further support for the hypothesis that CEQderived DNA adducts may play an important role in estrogen-mediated carcinogenesis. We propose that the above methodologies, with femtomole detection limits, can be used in future identification of CEQ-derived DNA adducts present in human breast tissue extracts. These preliminary analyses of the two major depurinating adducts formed by reaction of E2-3,4-Q with DNA have the scope to determine whether these two compounds can be detected in human tissues by low temperature phosphorescence. Of these two depurinating adducts, the 4-OHE1(E2)-1-N3Ade is the only one that generates mutations leading to initiation of cancer (6, 59). Therefore, it is the most important as a potential biomarker of susceptibility to cancer. To discover whether this hypothesis is correct, it will be necessary to continue


these experiments with breast tissue from women with and without cancer. The success of this approach depends on the formation of 4-OHE1(E2)-1-N3Ade in much larger amounts in women with breast cancer and undetectable or low amounts in tissue from women without cancer. This approach to developing biomarkers of susceptibility to cancer includes both depurinating DNA adducts and the conjugates of CE-3,4-Q with GSH and their derivatives, namely, Cys and NAcCys conjugates (26, 59).

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Acknowledgment. The Ames Laboratory is operated for the U.S. Department of Energy by Iowa State University under Contract No. W-7405-Eng-82. This work was supported by a grant from the National Cancer Institute (Program Project Grant 2PO1 CA49210-12). We also acknowledge partial support from the Office of Biological and Environmental Research, Office of Energy Research, U.S. Department of Energy.

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Spectral Characterization of Catechol Estrogen Quinone (CEQ

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