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 α-(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 Cu²⁺ 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.