gamma-Irradiation-induced DNA damage enhances NO production via NF-kappaB activation in RAW264.7 cells.
We investigated the mechanism of augmentation of nitric oxide (NO) production in the murine macrophage cell line RAW264.7 after gamma-irradiation. The cells treated with interferon-gamma (IFN-gamma) or lipopolysaccharide (LPS) showed enhanced NO production by gamma-irradiation in a dose-dependent manner, accompanying the induction of inducible nitric oxide synthase (iNOS) expression. Nuclear factor kappa B (NF-kappaB) activation was induced 1 h after gamma-irradiation dose-dependently, which was detected by the degradation of I-kappaB. Inhibitors of I-kappaB degradation, MG132 and N(alpha)-p-tosyl-L-lysine chloromethyl ketone (TLCK), suppressed the further increase by gamma-irradiation in IFN-gamma-induced NO production, showing that gamma-irradiation induced NO production via NF-kappaB activation. Although NF-kappaB 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(2)O(2) showed only slight enhancement of IFN-gamma-induced NO production. The DNA damaging agents camptothecin and etoposide enhanced IFN-gamma-induced NO production and showed I-kappaB 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 gamma-irradiation in IFN-gamma-induced NO production and the I-kappaB degradation by gamma-irradiation. We concluded that (1) the increase in NO production was due to direct DNA damage by gamma-irradiation, and that (2) PARP activation through DNA damage induced NF-kappaB activation, leading to iNOS expression and NO production.
Ibuki
Yuko
Y
Laboratory of Radiation Biology, Graduate School of Nutritional and Environmental Sciences, University of Shizuoka, 52-1, Yada, 422-8526, Shizuoka, Japan.
Mizuno
Sumio
S
Goto
Rensuke
R
D000818:Animals; D002460:Cell Line; D004249:DNA Damage; D004789:Enzyme Activation; D005720:Gamma Rays; D015151:Immunoblotting; D007371:Interferon-gamma; D008070:Lipopolysaccharides; D051379:Mice; D016328:NF-kappa B / Q000037:antagonists & inhibitors / Q000096:biosynthesis; D009569:Nitric Oxide / Q000037:antagonists & inhibitors / Q000096:biosynthesis*; D019001:Nitric Oxide Synthase / Q000096:biosynthesis; D052247:Nitric Oxide Synthase Type II; D011065:Poly(ADP-ribose) Polymerases / Q000378:metabolism; D015398:Signal Transduction; D013997:Time Factors
D008070:Lipopolysaccharides; D016328:NF-kappa B; D009569:Nitric Oxide; D007371:Interferon-gamma; D019001:Nitric Oxide Synthase; D052247:Nitric Oxide Synthase Type II; C496317:Nos2 protein, mouse; D011065:Poly(ADP-ribose) Polymerases