Estrogens, including the natural hormones estrone (E1) and estradiol (E2), are thought to be involved in tumor induction. Specifically, catechol estrogen quinones (CEQs) derived from the catechol estrogens 4-hydroxyestrone (4-OHE1) and 4-hydroxyestradiol (4-OHE2) react with DNA and form DNA adducts (Cavalieri, E. L., et al. (1997) Proc. Natl Acad. Sci. U.S.A. 94, 10037). CEQs are also conjugated with GSH, a reaction that prevents damage to DNA, providing biomarkers of exposure to CEQs. Current detection limits for these analytes by HPLC with multichannel electrochemical detection are in the picomole range (Devanesan, P., et al. (2001) Carcinogenesis 22, 489). To improve the detection limit of CEQ-derived conjugates, spectrophotometric monitoring was investigated. Fluorescence and/or phosphorescence spectra of the 4-OHE1, 4-OHE2, Cys, N-acetylcysteine (NAcCys), 4-OHE1-2-SG, and 4-OHE2-2-SG conjugates and their decomposition products 4-OHE1-2-NAcCys and 4-OHE2-2-NAcCys were obtained at 300 and 77 K. It is shown that (i) 4-OHE1- and 4-OHE2-derived SG and NAcCys conjugates are weakly fluorescent at 300 K (with the emission maximum at 332 nm) but strongly phosphorescent at 77 K; (ii) Cys and NAcCys exhibit fluorescence and phosphorescence only at 77 K; and (iii) 4-OHE1 and 4-OHE2 are weakly fluorescent at 300 and 77 K and not phosphorescent. The phosphorescence spectra of SG and NAcCys conjugates are characterized by a weak origin band at ∼383 nm and two intense vibronic bands at 407 and 425 nm. After they are cooled from 300 to 77 K, the total luminescence intensity of SG and NAcCys conjugates increases by a factor of ∼150 predominantly due to phosphorescence enhancement. 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. The concentration LOD is approximately 10-9 M. Therefore, we propose that capillary electrophoresis interfaced with low temperature phosphorescence detection can be used to test for human exposure to CEQs by analyzing urine.
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