Hepatocellular oxidative DNA injury induced by macrophage-derived nitric oxide

Previous studies have indicated that splenic macrophages migrate into the liver and play a role in endotoxin-induced hepatic damage. The present study was designed to elucidate the mechanisms of hepatocyte injury induced by activated splenic macrophages, focusing especially on endogenously released NO and oxidative DNA alterations in hepatocytes. Splenic macrophages isolated from Wistar rats were incubated with either lipopolysaccharide (LPS) or interferon-γ (IFN-γ) and cocultured with hepatocytes. Nitrite and nitrate levels in the culture medium were measured, and inducible-type NO synthase (iNOS) and nitrotyrosine were determined by immunofluorescence staining. The ratio of 8-hydroxy-deoxyguanosine (8-OH-dG) to deoxyguanosine (dG) was measured by high-performance liquid chromatography, and single-stranded DNA in hepatocytes was detected with acridine orange. NO release and nitrotyrosine expression in hepatocytes increased after 8 h of coculture with activated macrophages, and this coculture also induced increases in the 8-OH-dG/dG ratio and single-stranded DNA in the hepatocytes. These alterations were attenuated by superoxide dismutase (SOD) and NO synthesis inhibitors. A similar pattern of alterations was observed in hepatocytes incubated with SIN-1, and these changes were also prevented by SOD. These results suggest that activated macrophage-derived NO and its oxidative metabolite, peroxynitrite, play key roles in hepatocyte injury during inflammation, and cause subsequent DNA damage in surviving hepatocytes.