MUTAGENESIS INDUCED BY INTRACELLULAR PRODUCED HOCL IN AIRWAY EPITHELIAL CELLS

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

ORLANDO, FL

Congreso; 17TH ANNUAL MEETING OF THE SFRBM; 2010

MUTAGENESIS INDUCED BY INTRACELLULARLY PRODUCED HOCL IN AIRWAY EPITHELIAL CELLS SANDRA E GOMEZ-MEJIBA1,2, MARIA S GIMENEZ2, ZILI ZHAI1, and DARIO C RAMIREZ11,2, MARIA S GIMENEZ2, ZILI ZHAI1, and DARIO C RAMIREZ11 1OMRF, Oklahoma City, OK, USA, 2IMIBIO-SL, UNSL, SAN LUIS, ARGENTINAOMRF, Oklahoma City, OK, USA, 2IMIBIO-SL, UNSL, SAN LUIS, ARGENTINA, MARIA S GIMENEZ2, ZILI ZHAI1, and DARIO C RAMIREZ11 1OMRF, Oklahoma City, OK, USA, 2IMIBIO-SL, UNSL, SAN LUIS, ARGENTINAOMRF, Oklahoma City, OK, USA, 2IMIBIO-SL, UNSL, SAN LUIS, ARGENTINA Neutrophil infiltration is thought to be a key event in lung carcinogenesis induced by acute irritation of the airways. Myeloperoxidase (MPO) is present in the azurolphil’s granules of neutrophils and released upon activation. During acute injury of the lung by endotoxin, infiltrating and activated neutrophils release a large amount of MPO in the airways. Myeloperoxidase is the only mammalian enzyme that produces HOCl at physiological pH. Herein we sought to tests whether intracellularly produced HOCl causes genotoxicity and affect the repair mechanisms in airway epithelial cells that lead to mutations. Upon co-incubation 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-oxodG, and mutations in the hypoxanthine-guanine phosporibosyltransferase (HPRT) gene. We determined DNAcentered radicals by trapping them in situ and in real time with DMPO to form DNA-DMPO nitrone adducts and thus further oxidation and mutagenicity is prevented. We determined the frequency of mutation of the HPRT gene by selecting cells resistant to 6-thioguanine. Cells with a mutation in this gene survive in medium containing 6-TG, whereas non-mutated cells die. We determined uptake of MPO released form neutrophils and intracellularly produced HOCl using immunochemistry and aminophenyl fluorescein (APF), respectively. Inactivated MPO did not produce APF fluorescence, DNA-centered radicals, 8- oxo-dG or mutations in A549 cells. APF 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-oxodG, increased nitrone adducts and blocked HPRT gene mutation. HPRT gene mutation was due to intracellularly produced HOCl but not HOCl produced by extracellular MPO. Our findings establish the critical role of the transient formation of DNA-centered radical during mutagenesis produced by HOCl generated inside epithelial cells that might explain neutrophilic inflammation-induced lung carcinogenesis. Inhibitors of MPO, cell permeable scavengers of HOCl or spin traps may help prevent this process. Supported by NIEHS 5R00ES015415-04.(HPRT) gene. We determined DNAcentered radicals by trapping them in situ and in real time with DMPO to form DNA-DMPO nitrone adducts and thus further oxidation and mutagenicity is prevented. We determined the frequency of mutation of the HPRT gene by selecting cells resistant to 6-thioguanine. Cells with a mutation in this gene survive in medium containing 6-TG, whereas non-mutated cells die. We determined uptake of MPO released form neutrophils and intracellularly produced HOCl using immunochemistry and aminophenyl fluorescein (APF), respectively. Inactivated MPO did not produce APF fluorescence, DNA-centered radicals, 8- oxo-dG or mutations in A549 cells. APF 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-oxodG, increased nitrone adducts and blocked HPRT gene mutation. HPRT gene mutation was due to intracellularly produced HOCl but not HOCl produced by extracellular MPO. Our findings establish the critical role of the transient formation of DNA-centered radical during mutagenesis produced by HOCl generated inside epithelial cells that might explain neutrophilic inflammation-induced lung carcinogenesis. Inhibitors of MPO, cell permeable scavengers of HOCl or spin traps may help prevent this process. Supported by NIEHS 5R00ES015415-04.HPRT gene by selecting cells resistant to 6-thioguanine. Cells with a mutation in this gene survive in medium containing 6-TG, whereas non-mutated cells die. We determined uptake of MPO released form neutrophils and intracellularly produced HOCl using immunochemistry and aminophenyl fluorescein (APF), respectively. Inactivated MPO did not produce APF fluorescence, DNA-centered radicals, 8- oxo-dG or mutations in A549 cells. APF 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-oxodG, increased nitrone adducts and blocked HPRT gene mutation. HPRT gene mutation was due to intracellularly produced HOCl but not HOCl produced by extracellular MPO. Our findings establish the critical role of the transient formation of DNA-centered radical during mutagenesis produced by HOCl generated inside epithelial cells that might explain neutrophilic inflammation-induced lung carcinogenesis. Inhibitors of MPO, cell permeable scavengers of HOCl or spin traps may help prevent this process. Supported by NIEHS 5R00ES015415-04.HPRT gene mutation. HPRT gene mutation was due to intracellularly produced HOCl but not HOCl produced by extracellular MPO. Our findings establish the critical role of the transient formation of DNA-centered radical during mutagenesis produced by HOCl generated inside epithelial cells that might explain neutrophilic inflammation-induced lung carcinogenesis. Inhibitors of MPO, cell permeable scavengers of HOCl or spin traps may help prevent this process. Supported by NIEHS 5R00ES015415-04.