Formation of Two Novel Estrogen Guanine Adducts and HPLC/MS Detection of 4-Hydroxyestradiol-N7-Guanine in Human Urine

• Published in: Chemical research in toxicology 2008-08, Vol.21 (8), p.1622-1630
• Main Authors: BRANSFIELD, Leslie A., RENNIE, Alissa, VISVANATHAN, Kala, ODWIN, Shelly-Ann, KENSLER, Thomas W., YAGER, James D., FRIESEN, Marlin D., GROOPMAN, John D.
• Format: Article
• Language: English
• Subjects: Biomarkers - chemistry; Biomarkers - urine; Chromatography, High Pressure Liquid; DNA Adducts - chemistry; DNA Adducts - metabolism; Estradiol - analogs & derivatives; Estradiol - chemistry; Estradiol - urine; Estrogens, Catechol; Female; Guanine - analogs & derivatives; Guanine - chemistry; Guanine - urine; Humans; Spectrometry, Mass, Electrospray Ionization
• ISSN: 0893-228X; 1520-5010
• DOI: 10.1021/tx800145w

Estrogen-DNA adducts are potential biomarkers for assessing the risk of developing of a number of hormonally modified cancers, including breast cancer. Formation of the 4-hydroxyestradiol-N(7)-guanine (4-OHE2-N(7)-guanine) adduct from the reaction of estradiol-3,4-quinone with DNA and its detection in vivo has been established. With the ultimate goal of exploring estrogen-DNA adducts as biomarkers in experimental and human investigations, the 4-OHE2-N(7)-guanine was synthesized, and preliminary studies demonstrated that this adduct was detectable in all 10 female human urine samples examined. Therefore, more extensive investigations were conducted to study this compound's chemical-physical properties and to examine the stability of 4-OHE2-N(7)-guanine under a range of pH conditions that might influence biomarker measurement. Under neutral to alkaline conditions, 4-OHE2-N(7)-guanine could completely oxidize to an 8-oxo-guanine derivative. This derivative was isolated by HPLC, and mass spectrometry confirmed the oxidized compound by demonstrating the formation of an m/ z 168 fragment, generated by oxygen addition to guanine. Furthermore, investigation of the 4-OHE2-N(7)-2'-deoxyguanosine nucleoside adduct showed that under alkaline conditions a formamidopyrimidine analogue was produced. The formamidopyrimidine derivative forms from ring opening of the guanine imidazole ring following C-8 oxidation in the N(7), N(9) disubstituted guanine. Formation of both of these oxidized estrogen-guanine DNA adducts has precedent with other chemical agents that covalently bind to the N(7) position in guanine. Therefore, the development and application of methods to measure estrogen-guanine adducts will need to also consider these new adducts, and the biological implications of these compounds will need to be explored to determine their contribution to estrogen toxicology.