Intracellular MPO and genomic damage in epithelial cells

DARIO C. RAMIREZ; MARIA S. GIMENEZ; SANDRA E. GOMEZ-MEJIBA

WASHINGTON DC

Congreso; EXPERIMENTAL BIOLOGY 2011; 2011

FASEB

INTRACELLULAR MPO AND GENOMIC DAMAGE IN EPITHELIAL CELLS DARIO C RAMIREZ1, SANDRA E GOMEZ-MEJIBA1 and MARIA S GIMENEZ21, SANDRA E GOMEZ-MEJIBA1 and MARIA S GIMENEZ2 1 EXPERIMENTAL THERAPEUTICS, OMRF, OKLAHOMA CITY, OKEXPERIMENTAL THERAPEUTICS, OMRF, OKLAHOMA CITY, OK 2 IMIBIO-SL, San Luis, ArgentinaIMIBIO-SL, San Luis, Argentina During acute injury of the lung by endotoxin, infiltrating and activated neutrophils release a large amount of MPO in the airways that causes genomic damage in epithelial cells. Herein we sought to tests whether intracellularly produced HOCl causes genotoxicity in airway epithelial cells that lead to mutations. Upon coincubation with activated human neutrophils released MPO is accumulated inside A549 airway epithelial cells. Exposure of these cells to a flow of H2O2, MPO produced HOCl inside the cell that causes DNA-centered radicals, 8-oxo-dG, and mutations in the hypoxanthine-guanine phosporibosyltransferase (HPRT) gene. Inactivated MPO did not produce HOCl-induced luminol fluorescence, DNAcentered radicals, 8-oxo-dG or mutations in A549 cells. Luminol fluorescence was prevented with cell permeable inhibitors of MPO activity and resveratrol—a cell permeable scavenger of HOCl, but not affected by DMPO. DMPO treatment resulted in reduced 8-oxo-dG, increased nitrone adducts and blockedHPRT gene mutation. We found that this mutation was due to intracellularly produced HOCl, but not HOCl produced by extracellular MPO. Mutagenesis produced by HOCl generated inside epithelial cells is a new mechanism of genomic damage caused at sites of neutrophil-mediated inflammation.2O2, MPO produced HOCl inside the cell that causes DNA-centered radicals, 8-oxo-dG, and mutations in the hypoxanthine-guanine phosporibosyltransferase (HPRT) gene. Inactivated MPO did not produce HOCl-induced luminol fluorescence, DNAcentered radicals, 8-oxo-dG or mutations in A549 cells. Luminol fluorescence was prevented with cell permeable inhibitors of MPO activity and resveratrol—a cell permeable scavenger of HOCl, but not affected by DMPO. DMPO treatment resulted in reduced 8-oxo-dG, increased nitrone adducts and blockedHPRT gene mutation. We found that this mutation was due to intracellularly produced HOCl, but not HOCl produced by extracellular MPO. Mutagenesis produced by HOCl generated inside epithelial cells is a new mechanism of genomic damage caused at sites of neutrophil-mediated inflammation.