Endogenous Oxidative Damage of Deoxycytidine in DNA

Three major oxidation products of 2'-deoxycytidine (dC)-5-hydroxy-2'-deoxycytidine (oh5dC), 5-hydroxy-2'-deoxyuridine (oh5dU), and 5,6-dihydroxy-5,6-dihydro-2'-deoxyuridine (dUg)-were analyzed from enzymatically hydrolyzed DNA with reversed-phase high-performance liquid chromatography coupled to electrochemical detection. oh5dC and oh5dU can be detected with high sensitivity (50 fmol) and selectivity (0-0.2 V) from hydrolyzed DNA. dUg is not electrochemically active but can be measured by dehydrating it into oh5dU. The quantities of oh5dC, dUg, and oh5dU in untreated commercial-grade calf thymus DNA are 10, 10, and 0.75 fmol/μ g of DNA, respectively. These levels increased substantially when calf thymus DNA was exposed to ionizing radiation, H2O2alone, H2O2and combinations of Fe3+or Cu2+and ascorbate, near-UV light (365 nm), near-UV light in the presence of menadione, and OsO4, indicating that oh5dC, oh5dU, and dUg are major oxidative DNA damage products. The steady-state levels of these products were determined from freshly extracted rat tissues and ranged from