γ-Irradiation-induced DNA damage enhances NO production via NF-κB activation in RAW264.7 cells

Abstract

We investigated the mechanism of augmentation of nitric oxide (NO) production in the murine macrophage cell line RAW264.7 after γ-irradiation. The cells treated with interferon-γ (IFN-γ) or lipopolysaccharide (LPS) showed enhanced NO production by γ-irradiation in a dose-dependent manner, accompanying the induction of inducible nitric oxide synthase (iNOS) expression. Nuclear factor kappa B (NF-κB) activation was induced 1 h after γ-irradiation dose-dependently, which was detected by the degradation of I-κB. Inhibitors of I-κB degradation, MG132 and N α- p-tosyl- l-lysine chloromethyl ketone (TLCK), suppressed the further increase by γ-irradiation in IFN-γ-induced NO production, showing that γ-irradiation induced NO production via NF-κB activation. Although NF-κB is known to be a redox-sensitive transcription factor, the antioxidant agents N-acetyl-cysteine (NAC) and 6-hydroxy-2,5,7,8-tetramethyl-chroman-2-carboxylic acid (trolox) showed no suppression and treatment with H 2O 2 showed only slight enhancement of IFN-γ-induced NO production. The DNA damaging agents camptothecin and etoposide enhanced IFN-γ-induced NO production and showed I-κB degradation, indicating that the increase in NO production was due to direct DNA damage. Furthermore, 3-aminobenzamide (3AB) and benzamide, inhibitors of poly (ADP-ribose) polymerase (PARP) that are activated upon recognition of DNA strand breaks, suppressed the further increase by γ-irradiation in IFN-γ-induced NO production and the I-κB degradation by γ-irradiation. We concluded that (1) the increase in NO production was due to direct DNA damage by γ-irradiation, and that (2) PARP activation through DNA damage induced NF-κB activation, leading to iNOS expression and NO production.