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Hepatocellular oxidative DNA injury induced by macrophage-derived nitric oxide
1Department of Internal Medicine, School of Medicine, Keio University, Tokyo, Japan.
Abstract:
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-gamma (IFN-gamma) 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.
Insights
Activated splenic macrophages release nitric oxide (NO), causing DNA damage in liver cells. This damage, linked to inflammation, can be reduced by inhibiting NO synthesis and using antioxidants like superoxide dismutase (SOD).
Area of Science:
- Immunology
- Hepatology
- Molecular Biology
Background:
- Splenic macrophages are known to infiltrate the liver and contribute to endotoxin-induced hepatic damage.
- The precise mechanisms by which activated splenic macrophages induce hepatocyte injury require further elucidation.
Purpose of the Study:
- To investigate the role of endogenous nitric oxide (NO) and oxidative DNA alterations in hepatocyte injury induced by activated splenic macrophages.
- To understand the contribution of peroxynitrite, an oxidative metabolite of NO, to DNA damage in hepatocytes.
Main Methods:
- Co-culture of Wistar rat splenic macrophages (activated with lipopolysaccharide or interferon-gamma) with hepatocytes.
- Measurement of nitrite and nitrate levels, immunofluorescence staining for inducible-type NO synthase (iNOS) and nitrotyrosine.
- Quantification of 8-hydroxy-deoxyguanosine (8-OH-dG) to deoxyguanosine (dG) ratio and detection of single-stranded DNA in hepatocytes.
- Assessment of the effects of superoxide dismutase (SOD) and NO synthesis inhibitors.
Main Results:
- Activated macrophages significantly increased NO release and nitrotyrosine expression in hepatocytes.
- Coculture led to elevated 8-OH-dG/dG ratios and single-stranded DNA in hepatocytes, indicating DNA damage.
- These detrimental effects were significantly reduced by SOD and NO synthesis inhibitors.
- Similar DNA damage was observed with SIN-1 treatment and prevented by SOD.
Conclusions:
- Activated macrophage-derived NO and peroxynitrite are key mediators of hepatocyte injury and DNA damage during inflammation.
- Oxidative stress induced by macrophages contributes to the pathogenesis of liver damage.
- Targeting NO production and oxidative stress may offer therapeutic strategies for inflammatory liver diseases.