Myeloperoxidase-dependent oxidatively generated modification of the genome: a possible link between inflammation and carcinogenesis

SANDRA E. GOMEZ-MEJIBA; JAYA CHILAKAPATI; REBECCA FARIS; KIRK KITCHIN; RONALD P. MASON; DARIO C. RAMIREZ

Chapel Hill, NC

Encuentro; The 6th International Human Peroxidase Meeting; 2009

Cleveland Clinic

Myeloperoxidase-dependent oxidatively generated modification of the genome. A possible link between inflammation and carcinogenesis Sandra E. Gomez-Mejiba; Zhai, Zili, Rebecca Faris, Ronald P. Mason, and Dario C. Ramirez Free Radical Biology and Aging Research Program, Oklahoma Medical Research Foundation. Oklahoma City, OK 73104; Laboratory of Pharmacology, National Institute of Environmental Health Sciences, RTP, NC 27709 Infiltration of neutrophils into irritated tissues and oxidatively generated damage to the genomic DNA has been suggested to be the link between inflammation and carcinogenesis, however the mechanism remains unclear. Some of the myeloperoxidase contained in neutrophils or its model human-leukemia (HL)-60 cells is located in close proximity to the genomic DNA. MPO is also released with DNA during neutrophil extracellular traps. During activation of neutrophils H2O2 is produced and MPO uses it to produce hypochlorous acid (HOCl). HOCl is a strong oxidant that reacts with nucleic acid bases and sugars producing chloramines that decompose forming nitrogen- and carbon-centered radicals. We hypothesized that HOCl at concentrations similar to those found in site of neutrophil infiltration and activation produce DNA-centered radicals that we can trap with the nitrone 5,5-dimethyl-1-pyrroline N-oxide (DMPO). DMPO diffuses throughout all cell compartments and traps and prevents oxidative fragmentation of the DNA. DMPO binds covalently to the DNA and forms radical adducts that then decay forming nitrone adducts. The detection of nitrone adducts is performed using an antibody anti-DMPO and immunoassays. We sought to study DNA-centered radicals induced during the chlorinating cycle of MPO that uses low concentrations of H2O2 (mM) and physiological concentrations of chloride. We used our immuno-spin trapping assay to show experimental conditions to detect calf-thymus DNA-centered radicals induced by HOCl, MPO/H2O2 (glucose-glocuse oxidase)/Cl or formed DNA radicals in HL-60 cells. We found that DMPO traps DNA-centered radicals generated by HOCl, MPO and low concentrations of H2O2. We also found that DNA-centered radicals inside HL-60 cells that colocalize with genomic DNA and myeloperoxidase. MPO-derived nitrone adduct formation was prevented by scavengers of HOCl, inhibitors of MPO or scavengers of H2O2. The information presented suggests that HOCl produced by neutrophils is an important mechanism of oxidatively-induced modification to the genome under inflammatory conditions that may explain how inflammation initiates carcinogenesis.