Characterization and investigation of 4-hydroxyestrogen-guanine adducts as potential biomarkers of breast cancer risk

Estrogen-guanine adducts are potential biomarkers of breast cancer risk. These DNA adducts are formed through estrogen metabolism, in particular, through formation of estrogen quinones. Estrogen quinones are attacked by nucleophiles including DNA bases to form either stable or depurinating adducts. The depurinating adducts create mutagenic abasic sites in DNA and have been detected in tissues and urines of animals and humans.;Findings reported in this work include synthesis and mass spectral characterization of estrogen-guanine adducts and their isotopically labeled analogues. The adduct standards were used to understand their physical chemical stability and were also used to develop an isotope dilution mass spectrometric method to isolate and measure these compounds in biological samples. During structural characterization of the guanine adducts, two novel adduct analogues, an 8-oxo and a formamidopyrimidine derivative were identified. These derivatives were formed as reaction products under basic conditions.;The adduct standards were employed in the development of an isolation method that utilized multiple solid phase extraction columns as well as a derivatization step. The goal of the method development was its application to human samples to gain insight on the use of estrogen-guanine adducts as biomarkers of breast cancer risk. This method permitted the detection of estrogen-guanine adducts in the picogram range from both human and rat urine. Ten human urine samples were analyzed and adduct was detected in all samples validating the application of the method. Additionally, two rats strains, the estrogen sensitive ACI and the less sensitive SD rats were used to determine if there was a strain difference in adduct level following chronic estradiol treatment. There was only a borderline statistical difference between the two strains after three weeks of treatment whereas a 5-fold higher level of adducts were measured in the ACI rat urines compared to SD after six weeks of chronic dosing. The results described are encouraging and suggest there is an association between adduct level and cancer risk. It is necessary to continue work in both human and animal studies to determine if these adducts are in fact biomarkers of breast cancer and if they can be utilized for risk assessment.