ESR identification of free radicals formed from the oxidation of catechol estrogens by Cu2+.
Catechol estrogens are genotoxic, indirectly through redox cycling mechanisms leading to oxidative DNA damage and directly by formation of quinone-DNA adducts. Previously, we demonstrated that Cu2+ can oxidize estradiol (E2) catechols, establishing a copper redox cycle leading to the formation of DNA strand breaks. The goal of this study was to use electron spin resonance techniques to identify the free radical intermediates formed. The 2- and 4-OH catechols of E2 and ethinyl estradiol (EE) were oxidized to semiquinone intermediates, stabilized by Mg2+, when incubated with Cu2+. The 4-OH-EE semiquinone decayed more slowly than the 2-OH-EE semiquinone. Using the spin trap alpha-(4-pyridyl-1-oxide)-N-tert-butylnitrone, 4-OH-E2 plus Cu2+ generated hydroxyl radicals at a greater rate than 2-OH-E2 plus Cu2+. Formation of hydroxyl and methyl radical adducts was detected, using 5,5-dimethyl-1-pyrroline-N-oxide as the spin trap, when 2-OH-E2 was incubated with Cu2+ and 1% dimethyl sulfoxide. This was inhibited by the Cu1+ chelator bathocuproinedisulfonic acid and catalase. These data demonstrate that the oxidation of estrogen catechols by Cu2+ leads to a Cu-dependent mechanism of hydroxyl radical production via a hydrogen peroxide intermediate and suggest a mechanism for estrogen-associated site-specific DNA damage and mutagenesis.
Seacat
A M
AM
Department of Environmental Health Sciences, Johns Hopkins University School of Hygiene and Public Health, 615 N. Wolfe Street, Baltimore, Maryland 21205-2179, USA.
Kuppusamy
P
P
Zweier
J L
JL
Yager
J D
JD
D016227:Benzoquinones / Q000378:metabolism; D003300:Copper / Q000378:metabolism*; D003497:Cyclic N-Oxides / Q000378:metabolism; D004249:DNA Damage; D004578:Electron Spin Resonance Spectroscopy; D004958:Estradiol / Q000031:analogs & derivatives / Q000378:metabolism; D002393:Estrogens, Catechol / Q000378:metabolism*; D004997:Ethinyl Estradiol / Q000031:analogs & derivatives / Q000378:metabolism; D005609:Free Radicals / Q000378:metabolism; D005978:Glutathione / Q000494:pharmacology; D006861:Hydrogen Peroxide / Q000378:metabolism; D017665:Hydroxyl Radical / Q000378:metabolism*; D015394:Molecular Structure; D016296:Mutagenesis; D009589:Nitrogen Oxides / Q000378:metabolism; D010084:Oxidation-Reduction; D010618:Phenanthrolines / Q000378:metabolism; D011725:Pyridines; D013113:Spin Labels
D016227:Benzoquinones; D003497:Cyclic N-Oxides; D002393:Estrogens, Catechol; D005609:Free Radicals; D009589:Nitrogen Oxides; D010618:Phenanthrolines; D011725:Pyridines; D013113:Spin Labels; C043438:alpha-(4-pyridyl-1-oxide)-N-tert-butylnitrone; C025232:semiquinone radicals; D017665:Hydroxyl Radical; D004997:Ethinyl Estradiol; D004958:Estradiol; C017245:5,5-dimethyl-1-pyrroline-1-oxide; C028559:bathocuproine sulfonate; D003300:Copper; D006861:Hydrogen Peroxide; D005978:Glutathione