1 voteInflammation, DNA-centered radicals, and oxidative genotoxicity: The role of HOCl produced by myeloperoxidase in carcinogenesis

SANDRA E. GOMEZ-MEJIBA; ZILI ZHAI; DARIO C. RAMIREZ

Congreso; ANNUAL MEETING OF THE SFRBM; 2009

Myeloid cells (macrophages and neutrophils) infiltrate and synthesize myeloperoxidase (MPO) in sites of inflammation, producing gentotoxicity. In RAW 264.7 macrophages, bacterial lipopolysaccharide (LPS) induces superoxide radical anion, nuclear deformation (nuclear protuberances), MPO synthesis, biomolecule oxidation and cell death. “Freezing” LPS-triggered macrophage activation with the nitrone spin trap 5,5-dimethyl-1-pyrroline N-oxide (DMPO) prevented cell activation and death. Oxidation of proteins and genomic DNA was also blocked, with formation of protein- and DNA-DMPO nitrone adducts, as analyzed by immuno-spin trapping with a polyclonal anti-DMPO serum. Interestingly, confocal microscopy analysis of these cells showed that MPO, genomic DNA, and DNA-DMPO nitrone adducts co-localized in the nuclear protuberances. These observations, and the fact that DNA is negatively charged and MPO is a cationic protein, suggest a role for uptaken or newly synthesized MPO in oxidative genotoxicity induced by myeloid cells in sites of inflammation. In order to understand MPO-induced formation of DNA-centered radicals, we studied DNA-DMPO nitrone adducts in calf thymus DNA treated with micromolar concentrations of hypochlorous acid (HOCl) added as a bolus or generated in situ by the MPO/H2O2/Cl- system in the presence of DMPO. We also investigated DNA-DMPO nitrone adducts inside living cells containing MPO. The cell models we used were: i) human leukemia (HL)-60 cells, which overexpress MPO, ii) RAW 264.7 macrophages activated with LPS (1 ng/ml for 24 h), to induce MPO, and iii) A549 human airway epithelial cells pre-loaded with human MPO. When these cells were activated with the phorbol ester PMA, the number of 6-thioguanine-resistant cells with the hypoxanthine-guanine phosphoribosyl transferase (HRPT) mutation increased. This mutation was prevented by each of the following: the NADPH oxidase inhibitor apocynin; the MPO inhibitors salicylhydroxamic acid and 4-aminobenzoic acid hydrazide; the cell-permeable HOCl scavenger resveratrol; and DMPO, which traps DNA-centered radicals and prevents further oxidation. Genomic DNA-centered radicals and further mutagenesis induced by activated myeloid cells in sites of inflammation can be prevented by blocking MPO activity, preventing formation of and/or scavenging HOCl, or trapping DNA-centered radicals. Our findings provide new therapeutic avenues for preventing carcinogenesis induced by infiltration and activation of myeloid cells in sites of inflammation, for example, in the lung exposed to particulate matter. SUPPORTED BY NIEHS 5R00ES015415-03