For many diseases, mediation of pathogenesis by nitric oxide (NO) has been suggested. In this study, we explored NO-induced viral pathogenesis with a focus on nucleic acid damage as evidenced by 8-nitroguanosine formation in vivo. Wild-type mice and littermate mice deficient in inducible NO synthase (iNOS) were infected with influenza or Sendai virus. Formation of 8-nitroguanosine in virusinfected lungs was assessed immunohistochemically with an antibody specific for 8-nitroguanosine. Extensive nitration of RNA either treated with peroxynitrite or obtained from cultured RAW 264 cells expressing iNOS was readily detected by this antibody. Strong 8-nitroguanosine immunostaining was evident primarily in the cytosol of bronchial and bronchiolar epithelial cells of virus-infected wild-type mice but not iNOS-deficient mice. This staining colocalized with iNOS immunostaining in the lung. 8-Nitroguanosine staining disappeared after addition of exogenous authentic 8-nitroguanosine during the antibody reaction and after pretreatment of tissues with sodium hydrosulfite, which reduces 8-nitroguanosine to 8-aminoguanosine. NO was generated in excess in lungs of wild-type mice but was eliminated in iNOSdeficient mice after virus infection; this result also correlated well with formation of 8-nitroguanosine and 3-nitrotyrosine. One consequence of the lack of iNOS expression was marked improvement in histopathological changes in the lung and the lethality of the infection without effects on cytokine responses and viral clearance. It is intriguing that 8-nitroguanosine markedly stimulated superoxide generation from cytochrome P450 reductase and iNOS in vitro. The present data constitute a demonstration of 8-nitroguanosine formation in vivo and suggest a potential role for NO-induced nitrative stress in viral pathogenesis.

Induction of haem oxygenase-1 (HO-1) may provide an important protective effect for cells against oxidative stress. Here, we investigated the mechanism of cytoprotection of HO-1 in solid tumour with a focus on the antiapoptotic activity of HO-1. Treatment of rat hepatoma AH136B cells with the HO inhibitor zinc protoporphyrin IX (ZnPP IX) or tin protoporphyrin IX resulted in extensive apoptotic changes of tumour cells both in vivo and in vitro. Caspase-3 activity of the ZnPP IX-treated hepatoma cells increased significantly. Moreover, ZnPP IX-induced apoptosis was completely inhibited by simultaneous incubation with a specific caspase-3 inhibitor and was partially abrogated by bilirubin, a reaction product of HO. In vivo ZnPP IX treatment did not affect nitric oxide (NO) production and tumour blood flow. Western blot analyses showed that HO-1 expression in AH136B cells was strongly upregulated by NO donors, for example, S-nitroso-N-acetyl penicillamine and propylamine NONOate in vitro; conversely, it was remarkably reduced in vivo by pharmacological blockade of NOS. We conclude that HO-1 may function in antiapoptotic defense of the tumour, and thus it may have important protective and beneficial effects for tumour cells against oxidative stress induced by NO, which is produced in excess during solid tumour growth in vivo.

Host defense functions of nitric oxide (NO) are known for many bacterial infections. In this study, we investigated the antimicrobial effect of NO in murine salmonellosis by using inducible NO synthase (iNOS)-deficient mice infected with an avirulent or virulent Salmonella enterica serovar Typhimurium strain. All iNOS-deficient mice died of severe septicemia within 6 days after intraperitoneal injection with an avirulent strain (LT2) to which wild-type mice were highly resistant; 50% lethal doses (LD 50 s) of the LT2 strain for iNOS-deficient and wild-type mice were 30 CFU and 7 ؋ 10 4 CFU, respectively. Lack of NO production in iNOS-deficient mice was verified directly by electron spin resonance spectroscopy. Bacterial yields in liver and blood were much higher in iNOS-deficient mice than in wild-type mice throughout the course of infection. Very small amounts of a virulent strain of serovar Typhimurium (a clinical isolate, strain Gifu 12142; LD 50 , 50 CFU) given orally caused severe septicemia in iNOS-deficient animals; wild-type mice tolerated higher doses (LD 50 , 6 ؋ 10 2 CFU). Histopathology of livers from infected iNOS-deficient mice revealed extensive damage, such as diffuse hepatocellular apoptosis and increased neutrophil infiltration, but livers from infected wild-type mice showed a limited number of microabscesses, consisting of polymorphonuclear cells and macrophages and low levels of apoptotic change. The LT2 strain was much more susceptible to the bactericidal effect of peroxynitrite than the Gifu strain, suggesting that peroxynitrite resistance may contribute to Salmonella pathogenicity. These results indicate that NO has significant host defense functions in Salmonella infections not only because of its direct antimicrobial effect but also via cytoprotective actions for infected host cells, possibly through its antiapoptotic effect.

Matrix metalloproteinases (MMPs) are critical mediators of tissue remodeling. Inappropriate regulation of MMPs causes many pathological events, including microbial invasion and inflammatory tissue damage. Some of the bacterial exoproteinases can effectively activate pro-MMPs (inactive zymogens) via limited proteolysis around their autoinhibitory domains. In addition, overproduction of nitric oxide (NO) may contribute to respiratory inflammation via the formation of reactive nitrogen species (RNS). Several studies have identified regulatory properties of NO/RNS on biomolecules due to functional modification of their cysteine residues. In fact, NO/RNS can mediate activation and expression of MMPs, because RNS can interact with a cysteine switch in the autoinhibitory domain, thus converting proMMPs into their active forms without proteolysis. Many studies have indicated that NO/RNS can participate in expression of various genes that affect immune-inflammatory responses, including MMPs. Although NO in some cases upregulates MMPs, S -nitrosothiols downregulate MMP-9 expression by suppressing the NF-kappaB pathway. While microbial proteinases cause excessive activation of MMPs and contribute to microbial pathogenesis, NO/RNS may modulate expression and activation of MMPs as well as various inflammatory mediators, depending on the redox status at sites of inflammation. Therefore, appropriate regulation of MMPs may be of potential therapeutic value for various infections and inflammatory lung diseases.