NITRONA 5,5-DIMETHYL-1-PYRROLINE N-OXIDE PREVENTS GENOTOXICITY AT SITES OF NEUTROPHILIC INFLAMMATION

LOPEZ C M; RAMIREZ D C; GÓMEZ MEJIBA S E

SAN JUAN

Congreso; SBC; 2023

Neutrophilic inflammation (NI) is an important process of oxidative damage to macromolecules at sites of acute and chronic inflammation.Neutrophils are innate immune cells that express myeloperoxidase (MPO). This is the only enzyme that, at physiological conditions, canproduce hypochlorous acid (HOCl). HOCl is a highly reactive species that can rapidly oxidize any macromolecule. Oxidation of genomicDNA by HOCl can change its structure and function, resulting in mutagenesis, followed by cell transformation and cancer. However, thereis no evidence of the intermediacy of a DNA-centered radical in the HOCl-driven DNA oxidation and further mutagenesis process. Therefore,in this investigation, we aimed to test whether the oxidation of DNA is mediated by a DNA-centered radical species, and subsequently totest if by trapping DNA-centered radical with the nitrone spin trap 5,5-dimethyl-1-pyrroline N-oxide (DMPO), mutagenesis of a specificgene can be prevented. To accomplish our aim we used biochemical and in vitro models. We used a biochemical model in which HOCl wasadded as a bolus or generated by the MPO-H2O2-Cl¯ system in the absence or presence of DMPO. In the absence of DMPO, DNA-DMPOnitrone adducts were not observed, but instead, 8-oxo-deoxyguanosine (8-oxo-dGuo)—one of the multiple products of DNA oxidation byHOCl, was formed. Generation of HOCl, inside A549 epithelial cells pre-loaded with active human MPO and subsequently treated withH2O2 in phosphate buffer (PBS), was observed using luminol or APF-two fairly specific probes for HOCl. Under these conditions, 8-oxodGuo was formed. However, when DMPO was present, 8-oxo-dGuo was not found, but DNA-DMPO nitrone adducts were formed. Thesedata suggest that the formation of a DNA-centered radical precedes the formation of 8-oxo-dGuo when DNA genomic is oxidized by HOCl.Moreover, the hypoxanthine-guanine phosphoribosyltransferase (hrpt) gene is highly sensitive to mutagenesis. Hrpt-mutant cells can survivein a medium containing 6-thioguanine (6-TG). Incubation of MPO-preloaded A549 epithelial cells with H2O2 in PBS resulted in a largenumber of 6-TG-resistant cells. This effect was blocked when either DMPO, ABAH-a fairly specific inhibitor of MPO, or resveratrol—ascavenger of HOCl were added before H2O2 addition. These data suggest that by trapping DNA-centered radicals, 8-oxo-dG and furthermutation of the genome of bystander cells at sites of NI can be prevented.