Catechol estrogen conjugates and DNA adducts in the kidney of male Syrian golden hamsters treated with 4-hydroxyestradiol: potential biomarkers for estrogen-initiated cancer

Abstract

Formation of depurinating adducts by reaction of catechol estrogen-3,4-quinones with DNA was proposed to be a tumor initiating event by estrogens [E.L. Cavalieri et al. (1997) Proc. Natl Acad. Sci. USA, 94, 10937-10942]. Under estrogenic imbalance, oxidation of catechol estrogens to quinones may compete with their detoxification by protective enzymes. The quinones formed can be detoxified by reaction with glutathione (GSH) or can covalently bind to DNA. To provide more support for this hypothesis, we developed a method to identify and quantify GSH, cysteine (Cys) and N-acetylCys conjugates of 4-hydroxyestrogens (4-OHE) in the kidneys of male Syrian hamsters treated with 4-hydroxyestradiol (4-OHE2) by intraperitoneal injection. The highest level of conjugates was observed 1 h after treatment, and almost none was detected after 24 h. Dose-response studies indicated conjugate formation after treatment with 0.5 micromol of 4-OHE2/100 g body weight, and formation increased up to a treatment level of 12 micromol/100 g body weight. GSH, Cys and N-acetylCys conjugates of 4-OHE were identified in the picomole range by high-performance liquid chromatography (HPLC) with multichannel electrochemical detection and confirmed by HPLC/tandem mass spectrometry. Treatment of tissue homogenates with beta-glucuronidase/sulfatase at 37 degrees C for 6 h before extraction resulted in a 12- to 20-fold increase in Cys conjugates from picomole to nanomole levels. Similar enhancement was observed by just incubating the tissue at 37 degrees C for 6 h. Evidence for the 4-OHE-1-N7Gua depurinating adducts was obtained by mass spectrometry. We conclude that GSH and Cys conjugates of the 4-OHE and the 4-OHE-N7Gua adducts can be utilized as biomarkers to detect estrogenic imbalance and potential susceptibility to tumor initiation.

Metabolomic applications of electrochemistry/Mass spectrometry

“…Figure 3 is an overlay of a single EC detector channel (240 mV) for eight samples representing 20-fold lower metabolite levels. These signals are due to pg quantities of analyte on column and are consistent with reported 10 to 1000-fold lower limits of detection obtained with EC-Array when compared to photodiode array detection [19,40].…”

“…Several analytical techniques are useful for metabolomics including gas chromatography with mass spectrometry (GC-MS) [15,16], liquid chromatography with coulometric array electrochemistry [17][18][19][20][21] (LC-ECArray), LC-MS [22][23][24], and nuclear magnetic resonance spectroscopy (NMR) [5,8,10,25]. The qualitative characteristics of techniques, such as MS and NMR, provide essential data for chromatographic peak identification and purity, structural elucidation, and for normalizing multivariate data.…”

Pharmacogenetics and Regulation of Human CytochromeP450 1B1: Implications in Hormone-Mediated Tumor Metabolism and a Novel Target for Therapeutic Intervention

“…Cavalieri et al showed that 4-OHE 2 , when converted to its corresponding quinone and semiquinone metabolites, covalently binds purines in DNA, and this reaction results in the formation of abasic sites (91). Several studies have subsequently arisen that have shown that these abasic sites are present in vivo and contribute to carcinogenesis (94)(95)(96)(97)(98)(99). These studies have also shown that catechol estrogen quinones bind proteins within the cell, further contributing to carcinogenesis (94).…”

Ultrafiltration tandem mass spectrometry of estrogens for characterization of structure and affinity for human estrogen receptors

“…Although both LC-MS [15][16][17][18][19][20] and GC-MS [15,16,[21][22][23] have been used for the rapid and sensitive identification of estrogens and their metabolites, GC-MS usually requires derivatization which adds to the complexity of sample preparation, increases the time required per analysis, and might introduce artifacts. To eliminate the need for sample derivatization, we developed a method based on LC-MS-MS. From among the solution-phase ionization methods that have been reported for the mass spectrometric analysis of estrogens, such as fast atom bombardment (FAB) [24,25], atmospheric pressure chemical ionization (APCI) [15,19], and electrospray [15,16,20,25,26], we selected electro-spray ionization.…”