Trapping DNA Radicals With DMPO Reduces Hypochlorous Acid-Induced 8-oxo-7,8-dihydro-2'-deoxyguanosine and Mutagenesis in Lung Epithelial Cells

Abstract

Pulmonary neutrophilic inflammation (PNI) is the recruitment and activation of neutrophils in the microvasculature with the release of myeloperoxidase (MPO) in the airways. Bystander epithelial cells can take up MPO, where it can generate HOCl. HOCl can react with DNA, generating DNA radicals, which then decay to produce several mutagenic end-oxidation products, such as 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxo-dGuo). Herein, we aimed to test whether HOCl-induced DNA radicals precede DNA oxidation and mutagenesis in A549 human lung epithelial cells as an in vitro model that resembles PNI. Interestingly, by trapping HOCl-induced DNA radicals, the nitrone spin trap 5,5-dimethyl-1-pyrroline N-oxide (DMPO) blocks the formation of 8-oxo-dGuo and possibly other end-oxidation products, forming DNA-DMPO nitrone adducts. By preventing DNA oxidation, DMPO reduces the mutation of the hypoxanthine phosphoribosyl transferase (hrpt) gene, one of the genes most sensitive to oxidative damage. The transcription factor p53 is known as the master regulator of the cell response to genomic damage. By trapping DNA radicals, DMPO also blocks the translocation of p53 to the cell nucleus, suggesting that by trapping DNA radicals with DMPO, end-oxidation products are prevented, and the cell response to genomic damage is blunted. Trapping DNA radicals to reduce the accumulation of HOCl-induced mutagenic end-oxidation products will provide new therapeutic avenues to reduce genotoxic damage during PNI.

Keywords: 8-oxo-dGuo; DNA radical; DNA-DMPO nitrone adduct; cell model; hrpt gene mutation; pulmonary neutrophilic inflammation.

Figure 1

Calf-thymus DNA radicalization by the MPO/H₂O₂/Cl⁻ biochemical system and its measurement using immuno-spin trapping. ELISA for DNA-DMPO nitrone adducts formed when calf-thymus DNA was incubated with MPO in buffer containing physiological concentrations of Cl⁻, and HOCl formation started with H₂O₂. Potassium cyanide (KCN), 4-aminobenzoic acid hydrazide (ABAH), salicylhydroxamic acid (SHA), taurine, or methionine was added before starting HOCl formation with 50 μM H₂O₂. The data are shown as the mean values ± s.e.m. from three separate experiments performed in triplicate. Asterisks indicate p  < 0.05.

Figure 2

Coculture of A549 lung epithelial cells with PMA-activated neutrophils showing NETs in sequential planar images. (A) Scanning confocal microscopy images showing the localization of histone H2B (green), MPO (red), and genomic DNA (blue, DAPI) in A549 lung epithelial cells coincubated with neutrophils. The measurement bars represent 10 μM. (B) Same as A, but PMA was added to activated neutrophils to form NETs. The measurement bars represent 20 μM. (C) Eighteen z-stacks of planar images of the image marked with a yellow arrow in B. This image shows the interaction of a NET generated during neutrophil activation with one A549 cell. The measurement bars represent 5 μm. The images are representative of three individual experiments.

Figure 3

Intracellular production of HOCl by MPO inside A549 epithelial cells. (A) A representative laser confocal image showing the intracellular location of MPO inside A549 cells incubated without or with 10 nM purified human MPO. (B) Effects of intracellularly produced HOCl on cell viability (MTT reduction assay, closed squares) and GAPDH activity (closed triangles) in A549 cell homogenates. MPO-loaded A549 cells were incubated in HBSS⁺ containing 140 mM NaCl with different concentrations of H₂O₂ for 15 min. ⁣^∗ and # indicate p  < 0.05 with respect to the viability and GAPDH activity of the control (cells not treated with H₂O₂), respectively. (C) Luminol oxidation by intracellularly produced HOCl by active MPO. The cells were loaded with MPO and then incubated with luminol and DMPO, ABAH, Res or Tau. (D) Effect of the inactivation of MPO or scavenging of HOCl on luminol oxidation by intracellularly produced HOCl. Like C, but cells were preloaded with active (MPO) or preinactivated MPO (KCN-MPO and ATZ-MPO). (E) Effects of MPO inhibition or HOCl scavenging on DNA-DMPO adduct formation. MPO-preloaded A549 cells were incubated with DMPO, with or without an inhibitor of MPO (ABAH) or scavenger of HOCl (Tau or Res). DNA-DMPO nitrone adducts were measured by ELISA and referred to as DNA bound to the culture plate. In C–D, intracellular HOCl production was triggered by the addition of a bolus of 50 μM H₂O₂, and the vehicle was HBSS⁺ containing 140 mM NaCl. The data are presented as the mean values ± s.e.m. from four independent experiments. In panels C–E, an asterisk indicates p  < 0.05 with respect to cells not preloaded with MPO and incubated with vehicle (HBSS⁺ alone). a.u., arbitrary units.

Figure 4

DMPO prevents the accumulation of 8-oxo-dGuo induced by intracellularly produced HOCl. (A) Quantification of 8-oxo-dGuo and DMPO-nitrone adducts in DNA isolated from A549 cells in which HOCl was intracellularly generated in the absence or presence of DMPO. The intracellular generation of HOCl was triggered by a continuous flow of H₂O₂ by the Glu/GO (5.6 mM Glu/1 mIU mL⁻¹ GO) system. The reactions were stopped 30 min later by rinsing the cells with 100 μM resveratrol in Tris-buffered saline (pH 7.8), an amine buffer used to scavenge excess HOCl inside the cells. (B) Same as A, but the cells were loaded with native MPO or MPO that was previously inactivated (KCN-MPO or ATZ-MPO). Reactions were performed in either the presence or absence of 50 mM DMPO to measure DNA-DMPO nitrone adducts or 8-oxo-dGuo, respectively. The data are shown as the mean values ± s.e.m. from three independent experiments. The asterisk (⁣^∗) indicates a difference (p  < 0.05) in DNA-DMPO nitrone adducts with respect to none (A549 cells not preloaded with MPO and incubated in HBSS⁺ alone). The pound symbol (#) indicates a difference in 8-oxo-dGuo with respect to none (i.e., A549 cells not preloaded with MPO and incubated with HBSS⁺ alone).

Figure 5

The intracellular generation of HOCl leads to the nuclear accumulation of p53. (A) Western blot analysis of p53 in the cytosolic and nuclear fractions of A549 epithelial cells preloaded with native MPO and then treated with the 5.6 mM Glu/1 mIU/mL GO system for 15 min in the presence or absence of DMPO. (B) Densitometric analysis of the p53 band intensities in the nuclear fraction shown in A using the via ImageJ software. Relative band intensities with respect to MPO preloaded cells (MPO) are depicted. The data are representative images or mean values ± s.e.m. from three independent experiments. ⁣^∗p  < 0.05 and ⁣^∗∗p  < 0.001 between the groups being compared, as indicated by the segments. (C) Planar confocal image showing changes in the compartmentalization of p53 in A549 lung epithelial cells treated as described in A. A representative image of three separate experiments is shown. (D) In-plate trypan blue exclusion assay for measurement of 6-TG-resistant cells (mutant cells) after 14 days of culture (Supporting Information 1: Figure S4A). The data are presented as the means ± s.e.m. from four independent experiments performed in triplicate. Symbols indicate p  < 0.05. ⁣^∗ compares the different groups (white bars) with respect to A549 cells without MPO and treated with glucose (Glu) alone; # compares MPO-loaded A549 groups (black bars) with respect to cells preloaded with MPO and treated with Glu; and ^& compares groups of cells preloaded with or without MPO (black vs. white bars) for each treatment.

Figure 6

Scheme summarizing the main findings of this study. Pulmonary neutrophilic inflammation (PNI) in the lumen of airways causes the release of MPO. MPO is then taken up by airway epithelial cells. Inside airway epithelial cells, MPO produces HOCl, which upon reaction with genomic DNA causes DNA radical formation. DNA radicals quickly react with oxygen to give end-oxidation products, including 8-oxo-dGuo. 8-oxo-dGuo can lead to mutagenesis of the hrpt gene. The DNA damage-sensing mechanism is then activated by the translocation of p53 to the nucleus to trigger DNA repair mechanisms and determine cell fate. However, by trapping DNA radicals with DMPO, the formation of 8-oxodGuo, mutation of the hrpt gene, and p53 translocation into the nucleus are prevented.