UC Riverside

Endogenous and exogenous sources can induce reactive oxygen species (ROS), which can inflict damage to DNA. This damage has been linked to aging, cancer, and other human diseases. In this dissertation, we focus on several DNA lesions, which remain unexplored with regards to their induction by ROS via the Fenton-type reaction. We employed a highly sensitive and robust LC-MS/MS/MS coupled with stable isotope dilution method to aid us in the research at hand.

In Chapter 2, we investigated the induction of the (5'R) and (5'S) 8,5'-cyclopurine-2'-deoxynucleosides by ROS in vitro. Fenton-type reaction constitutes a relevant source of endogenous ROS since transition metal ions in the reduced state can react with H2O2, which is a byproduct of normal aerobic metabolism, to generate a highly reactive ·OH. Our results demonstrated that under increasing concentrations of Fenton-type reagents, both diastereomers cPu were generated in a dose-dependent manner, with the (5'R)-isomers being preferentially generated. This supported earlier findings that the (5'R) diastereomers of the purine cyclonucleosides are repaired more efficiently in mammalian cells than the (5'S) counterparts.

In Chapter 3, we described the accumulation of cPu in tissues of Long-Evan Cinnonmon rats, which bear a deletion in the Atp7b gene thus sharing many attributes of Wilson's disease. Wilson's disease entails defective excretion of copper ions into the bile, in which patients often suffer from hepatic, neurological and renal abnormalities following copper toxicosis. Therefore, this was a viable system to assess the induction of cPu from in vivo Fenton-type reactions. Our results showed that cPu accumulation occurred in age-dependent manner in liver and brain tissues.

In Chapter 4, we assessed the conversion of 5-methyl-2'-deoxycytidine (5-mdC) to its oxidative derivatives by Fenton-type reagents. A Tet/TDG-mediated pathway has been demonstrated to induce active cytosine demethylation in mammals through enzymatic oxidation and subsequent cleavage. Our results suggested ROS may induce demethylation without the use of Tet, potentially compromising the integrity of the genome and gene regulation. We showed that all three oxidation products of 5-mdC were induced in a dose-dependent manner.