Activated Leukocytes Oxidatively Damage DNA, RNA, and the Nucleotide Pool through Halide-Dependent Formation of Hydroxyl Radical

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Abstract
A variety of chronic inflammatory conditions are associated with an increased risk for the development of cancer. Because of the numerous links between DNA oxidative damage and carcinogenesis, a potential role for leukocyte-generated oxidants in these processes has been suggested. In the present study, we demonstrate a novel free transition metal ion-independent mechanism for hydroxyl radical (•OH)-mediated damage of cellular DNA, RNA, and cytosolic nucleotides by activated neutrophils and eosinophils. The mechanism involves reaction of peroxidase-generated hypohalous acid (HOCl or HOBr) with intracellular superoxide (O2•-) forming •OH, a reactive oxidant species implicated in carcinogenesis. Incubation of DNA with either isolated myeloperoxidase (MPO) or eosinophil peroxidase (EPO), plasma levels of halides (Cl- and Br-), and a cell-free O2•- -generating system resulted in DNA oxidative damage. Formation of 8-hydroxyguanine (8-OHG), a mutagenic base which is a marker for •OH-mediated DNA damage, required peroxidase and halides and occurred in the presence of transition metal chelators (DTPA ± desferrioxamine), and was inhibited by catalase, superoxide dismutase (SOD), and scavengers of hypohalous acids. Similarly, exposure of DNA to either neutrophils or eosinophils activated in media containing metal ion chelators resulted in 8-OHG formation through a pathway that was blocked by peroxidase inhibitors, hypohalous acid scavengers, and catalytically active (but not heat-inactivated) catalase and SOD. Formation of 8-OHG in target cells (HA1 fibroblasts) occurred in all guanyl nucleotide-containing pools examined following exposure to both a low continuous flux of HOCl (at sublethal doses, as assessed by [14C]adenine release and clonogenic survival), and hyperoxia (to enhance intracellular O2•- levels). Mitochondrial DNA, poly A RNA, and the cytosolic nucleotide pool were the primary targets for oxidation. Moreover, modest but statistically significant increases in the 8-OHG content of nuclear DNA were also noted. These results suggest that the peroxidase−H2O2−halide system of leukocytes is a potential mechanism contributing to the well-established link between chronic inflammation, DNA damage, and cancer development.