FORMATION AND CHARACTERIZATION OF A NOVEL 4-HYDROXYESTRADIOL-N7-GUANINE-FORMAMIDOPYRIMIDINE ADDUCT AND DETECTION OF 4-OHE2-N7-GUANINE IN HUMAN URINE
Leslie A. Bransfielda, Alissa Rennieb, Kala Visvanathanc, Shelly-Ann Odwinb, Thomas W. Kenslerb, James D. Yagerb, John D. Groopmanb
aJohns Hopkins University, Department of Chemistry, Baltimore, MD 21218
bJohns Hopkins University, Department of Environmental Health Sciences, Baltimore, MD 21205
cJohns Hopkins University, Department of Epidemiology, Baltimore, MD 21205
A major risk factor for breast cancer is the endogenous hormone, estrogen. Estrogen mediated carcinogenesis can involve its binding to estrogen receptors stimulating cell proliferation and its oxidative metabolism to catechols and then quinones which can form covalent adducts with DNA. Estrogen quinone-DNA adducts are also potential biomarkers for risk assessment of cancer development. The ability to quantitatively measure these adducts and assign a risk based on the levels of adducts could lead to earlier detection and possible prevention strategies. The estradiol (E2) quinone metabolites are electrophilic compounds that react with DNA to form both stable and depurinating adducts. The quinone derived from 4-hydroxyestradiol can react with DNA to form adenine and guanine adducts which depurinate from DNA. These initial N7-N9 doubly substituted E2 quinone-guanine adducts place a localized positive charge on the imidazole ring of the pyrimidine facilitating destabilization of the glycosidic bond resulting in depurination. Alternatively, the positive charge can also facilitate ring opening of the imidazole ring leading to a stable formamidopyrimidine (fapy) adduct. The in vitro half-life of the unstable 4-hydroxyestradiol-N7-deoxyguanosine (4-OHE2-N7-dG) adduct was reported to be 6.5 hrs which is sufficient for fapy production. We conducted studies to determine if 4-OHE2-N7-dG is converted to its fapy derivative (4-OHE2-fapy-dG). Analysis by mass spectrometry revealed an m/z of 572.4 which corresponds to 4-OHE2-fapy-dG. Upon ms/ms analysis, an m/z of 455.2 was found corresponding to loss of the deoxyribose moiety. Additional analysis by ms3 revealed fragments with m/z 167.9 and 140.1 corresponding to fapyG and loss of CO from fapyG respectively. These data confirm formation of 4-OHE2-fapy-dG in reactions involving the nucleoside. Further studies to investigate the possible formation of this adduct in calf thymus DNA and DNA from cultured rat hepatocytes treated with E2 are underway to determine if there is any biological significance to this novel estrogen adduct derivative. Confirmation of the fapy estrogen adduct in a biological system will add another piece to the puzzle in the complex metabolic profile of estrogen.
As mentioned, the estrogen quinones can react with DNA to form depurinating adducts which are potential biomarkers for risk assessment since they are a direct measure of DNA damage. Efforts have focused on developing a method to quantitatively measure E2-guanine adducts in human urine. Five human urine samples were processed by the developed method and 4-OHE2-N7-guanine was detected in all five samples with an average level of 92.1 ± 18.4 pg/mL with a range of 68-119.5 pg/mL.