Gaseous Nitric Oxide-Induced 8-Nitroguanine Formation in Human Lung Fibroblast Cells and Cell-Free DNA

Abstract

A time- and dose-dependent increase in 8-nitroguanine (8-NO2-G) was observed in human lung fibroblast cells (MRC-5) after treatment with gaseous NO-saturated buffer. It was also found that treatment with the inhibitor of inducible nitric oxide synthase (iNOS), NG-nitro-l-arginine methyl ester, significantly reduced the 8-NO2-G level in the gaseous NO-saturated buffer-treated MRC-5 cells. These results provide evidence indicating that NO gas causes DNA damage in mammalian cells, which involves the activation of iNOS and the subsequent generation of endogenous NO. On the other hand, a time- and dose-dependent increase in 8-NO2-G was also observed while DNA (isolated from MRC-5 cells) was incubated with gaseous NO-saturated buffer. These results suggest that part of the 8-NO2-G formation was due to direct modification of gaseous NO on DNA. Furthermore, an increase in nitrite concentration was found in both cell-free and MRC-5 cell-conditioned medium treated with gaseous NO-saturated buffer. Collectively, gaseous NO induced DNA damage by forming 8-NO2-G, a modification performed directly by the treated gaseous NO and indirectly by the following induction of endogenous NO. This effect might be an important pathway in genotoxicity of nitric oxides, and 8-NO2-G could act as a specific marker for DNA damage induced by gaseous NO, a common contaminatant in air pollution and cigarette smoke.