Nitric oxide in gastrointestinal epithelial cell carcinogenesis: linking inflammation to oncogenesis

Chronic inflammation of gastrointestinal tissues is a well-recognized risk factor for the development of epithelial cell-derived malignancies. Although the inflammatory mediators linking chronic inflammation to carcinogenesis are numerous, current information suggests that nitric oxide (NO) contributes to carcinogenesis during chronic inflammation. Inducible nitric oxide synthase (iNOS), expressed by both macrophages and epithelial cells during inflammation, generates the bioreactive molecule NO. In addition to causing DNA lesions, NO can directly interact with proteins by nitrosylation and nitosation reactions. The consequences of protein damage by NO appear to be procarcinogenic. For example, NO inhibits DNA repair enzymes such as human 8-oxodeoxyguanosine DNA glycosylase 1 and blocks apoptosis via nitrosylation of caspases. These cellular events permit DNA damage to accumulate, which is required for the numerous mutations necessary for development of invasive cancer. NO also promotes cancer progression by functioning as an angiogenesis factor. Strategies to inhibit NO generation during chronic inflammation or to scavenge reactive nitrogen species may prove useful in decreasing the risk of cancer development in chronic inflammatory gastrointestinal diseases.     

Oxidative and nitrosative events in asthma

Asthma affects over 15 million individuals in the United States, with over 1.5 million emergency room visits, 500,000 hospitalizations, and 5500 deaths each year, many of which are children. Airway inflammation is the proximate cause of the recurrent episodes of airflow limitation in asthma. Research applying molecular biology, chemistry, and cell biology to human asthma and model systems of asthma over the last decade has revealed that numerous biologically active proinflammatory mediators lead to increased production of reactive oxygen species (ROS) and the gaseous molecule nitric oxide (NO). Persistently increased ROS and NO in asthma lead to reactive nitrogen species (RNS) formation and subsequent oxidation and nitration of proteins, which may cause alterations in protein function that are biologically relevant to airway injury/inflammation. Eosinophil peroxidase and myeloperoxidase, leukocyte-derived enzymes, amplify oxidative events and are another enzymatic source of NO-derived oxidants and nitrotyrosine formation in asthma. Concomitant with increased generation of oxidative and nitrosative molecules in asthma, loss of protective antioxidant defense, specifically superoxide dismutase (SOD), contributes to the overall toxic environment of the asthmatic airway. This review discusses the rapidly accruing data linking oxidative and nitrosative events as critical participants in the acute and chronic inflammation of asthmatic airways.     

Nitrosation of uric acid induced by nitric oxide under aerobic conditions.

Uric acid is a well-established scavenger of reactive oxygen and nitrogen species such as hydroxyl radical and peroxynitrite. However, little attention has been paid to the relationship between uric acid and nitric oxide. This paper reports the identification and characterization of a reaction product of uric acid induced by nitric oxide. When uric acid was treated with nitric oxide gas in a neutral solution under aerobic conditions, uric acid was consumed, yielding an unknown product. The product was identified as nitrosated uric acid from mass spectrometric data, although the position of the nitroso group on the molecule was not determined. The nitrosated uric acid decomposed to several compounds including uric acid with a half-life of 2.2 min at pH 7.4 and 37 degrees C. The incubation of nitrosated uric acid with glutathione resulted in the formation of S-nitrosoglutathione. Nitrosated uric acid was also formed in the reaction with nitric oxide donors, but not with peroxynitrite. Nitrosated uric acid was detected in human serum and urine by in vitro treatment with a nitric oxide donor. In the reaction of glutathione with the nitric oxide donor, the addition of uric acid caused an increase in the yield of S-nitrosoglutathione. These results indicate that under aerobic conditions nitric oxide can convert uric acid into its nitroso derivative, which can give a nitroso group to glutathione. Uric acid may act as a vehicle of nitric oxide in humans.     

Reactive nitrogen species in the chemical biology of inflammation

The preponderance of epidemiological evidence now points to a strong association between chronic inflammation and cancers of several organs, including the gastrointestinal tract, liver, and lungs. The strongest evidence for a mechanistic link here involves the generation of reactive oxygen and nitrogen species by macrophages and neutrophils that respond to cytokines and other signaling processes arising at sites of inflammation. These reactive species cause oxidation, nitration, halogenation, and deamination of biomolecules of all types, including lipids, proteins, carbohydrates, and nucleic acids, with the formation of toxic and mutagenic products. This review, in honor of Bruce Ames, will focus on recent advances in our understanding of the protein and DNA damage caused by reactive nitrogen species produced by macrophages and neutrophils, with emphasis on nitric oxide, nitrous anhydride, peroxynitrite, and nitrogen dioxide radical.     

NO Synthase and NO-Dependent Signal Pathways in Brain Aging and Neurodegenerative Disorders: The Role of Oxidant/Antioxidant Balance

Nitric oxide and other reactive nitrogen species appear to play several crucial roles in the brain. These include physiological processes such as neuromodulation, neurotransmission and synaptic plasticity, and pathological processes such as neurodegeneration and neuroinflammation. There is increasing evidence that glial cells in the central nervous system can produce nitric oxide in vivo in response to stimulation by cytokines and that this production is mediated by the inducible isoform of nitric oxide synthase. Although the etiology and pathogenesis of the major neurodegenerative and neuroinflammatory disorders (Alzheimer's disease, amyothrophic lateral sclerosis, Parkinson's disease, Huntington's disease and multiple sclerosis) are unknown, numerous recent studies strongly suggest that reactive nitrogen species play an important role. Furthermore, these species are probably involved in brain damage following ischemia and reperfusion, Down's syndrome and mitochondrial encephalopathies. Recent evidence also indicates the importance of cytoprotective proteins such as heat shock proteins (HSPs) which appear to be critically involved in protection from nitrosative and oxidative stress. In this review, evidence for the involvement of nitrosative stress in the pathogenesis of the major neurodegenerative/ neuroinflammatory diseases and the mechanisms operating in brain as a response to imbalance in the oxidant/antioxidant status are discussed.     

Role of reactive oxygen and nitrogen species in the vascular responses to inflammation

Inflammation is a complex and potentially life-threatening condition that involves the participation of a variety of chemical mediators, signaling pathways, and cell types. The microcirculation, which is critical for the initiation and perpetuation of an inflammatory response, exhibits several characteristic functional and structural changes in response to inflammation. These include vasomotor dysfunction (impaired vessel dilation and constriction), the adhesion and transendothelial migration of leukocytes, endothelial barrier dysfunction (increased vascular permeability), blood vessel proliferation (angiogenesis), and enhanced thrombus formation. These diverse responses of the microvasculature largely reflect the endothelial cell dysfunction that accompanies inflammation and the central role of these cells in modulating processes as varied as blood flow regulation, angiogenesis, and thrombogenesis. The importance of endothelial cells in inflammation-induced vascular dysfunction is also predicated on the ability of these cells to produce and respond to reactive oxygen and nitrogen species. Inflammation seems to upset the balance between nitric oxide and superoxide within (and surrounding) endothelial cells, which is necessary for normal vessel function. This review is focused on defining the molecular targets in the vessel wall that interact with reactive oxygen species and nitric oxide to produce the characteristic functional and structural changes that occur in response to inflammation. This analysis of the literature is consistent with the view that reactive oxygen and nitrogen species contribute significantly to the diverse vascular responses in inflammation and supports efforts that are directed at targeting these highly reactive species to maintain normal vascular health in pathological conditions that are associated with acute or chronic inflammation.     

Human atherosclerotic intima and blood of patients with established coronary artery disease contain high density lipoprotein damaged by reactive nitrogen species

High density lipoprotein (HDL) is the major carrier of lipid hydroperoxides in plasma, but it is not yet established whether HDL proteins are damaged by reactive nitrogen species in the circulation or artery wall. One pathway that generates such species involves myeloperoxidase (MPO), a major constituent of artery wall macrophages. Another pathway involves peroxynitrite, a potent oxidant generated in the reaction of nitric oxide with superoxide. Both MPO and peroxynitrite produce 3-nitrotyrosine in vitro. To investigate the involvement of reactive nitrogen species in atherogenesis, we quantified 3-nitrotyrosine levels in HDL in vivo. The mean level of 3-nitrotyrosine in HDL isolated from human aortic atherosclerotic intima was 6-fold higher (619 +/- 178 micromol/mol Tyr) than that in circulating HDL (104 +/- 11 micromol/mol Tyr; p < 0.01). Immunohistochemical studies demonstrated striking colocalization of MPO with epitopes reactive with an antibody to 3-nitrotyrosine. However, there was no significant correlation between the levels of 3-chlorotyrosine, a specific product of MPO, and those of 3-nitrotyrosine in lesion HDL. We also detected 3-nitrotyrosine in circulating HDL, and linear regression analysis demonstrated a strong correlation between the levels of 3-chlorotyrosine and levels of 3-nitrotyrosine. These observations suggest that MPO promotes the formation of 3-chlorotyrosine and 3-nitrotyrosine in circulating HDL but that other pathways also produce 3-nitrotyrosine in atherosclerotic tissue. Levels of HDL isolated from plasma of patients with established coronary artery disease contained twice as much 3-nitrotyrosine as HDL from plasma of healthy subjects, suggesting that nitrated HDL might be a marker for clinically significant vascular disease. The detection of 3-nitrotyrosine in HDL raises the possibility that reactive nitrogen species derived from nitric oxide might promote atherogenesis. Thus, nitrated HDL might represent a previously unsuspected link between nitrosative stress, atherosclerosis, and inflammation.     

The role of reactive nitrogen species in secondary spinal cord injury: formation of nitric oxide, peroxynitrite, and nitrated protein

To determine whether reactive nitrogen species contribute to secondary damage in CNS injury, the time courses of nitric oxide, peroxynitrite, and nitrotyrosine production were measured following impact injury to the rat spinal cord. The concentration of nitric oxide measured by a nitric oxide-selective electrode dramatically increased immediately following injury and then quickly declined. Nitro-L-arginine reduced nitric oxide production. The extracellular concentration of peroxynitrite, measured by perfusing tyrosine through a microdialysis fiber into the cord and quantifying nitrotyrosine in the microdialysates, significantly increased after injury to 3.5 times the basal level, and superoxide dismutase and nitro-L-arginine completely blocked peroxynitrite production. Tyrosine nitration examined immunohistochemically significantly increased at 12 and 24 h postinjury, but not in sham-control sections. Mn(III) tetrakis(4-benzoic acid)-porphyrin (a novel cell-permeable superoxide dismutase mimetic) and nitro-L-arginine significantly reduced the numbers of nitrotyrosine-positive cells. Protein-bound nitrotyrosine was significantly higher in the injured tissue than in the sham-operated controls. These results demonstrate that traumatic injury increases nitric oxide and peroxynitrite production, thereby nitrating tyrosine, including protein-bound tyrosine. Together with our previous report that trauma increases superoxide, our results suggest that reactive nitrogen species cause secondary damage by nitrating protein through the pathway superoxide + nitric oxide peroxynitrite protein nitration.     

Measurement and meaning of markers of reactive species of oxygen, nitrogen and sulfur in healthy human subjects and patients with inflammatory joint disease

Reactive species of oxygen, nitrogen and sulfur play cell signalling roles in human health, e.g. recent studies have shown that increased dietary nitrate, which is a source of RNS (reactive nitrogen species), lowers resting blood pressure and the oxygen cost of exercise. In such studies, plasma nitrite and nitrate are readily determined by chemiluminescence. At sites of inflammation, such as the joints of RA (rheumatoid arthritis) patients, the generation of ROS (reactive oxygen species) and RNS overwhelms antioxidant defences and one consequence is oxidative/nitrative damage to proteins. For example, in the inflamed joint, increased RNS-mediated protein damage has been detected in the form of a biomarker, 3-nitrotyrosine, by immunohistochemistry, Western blotting, ELISAs and MS. In addition to NO?, another cell-signalling gas produced in the inflamed joint is H2S (hydrogen sulfide), an RSS (reactive sulfur species). This gas is generated by inflammatory induction of H2S-synthesizing enzymes. Using zinc-trap spectrophotometry, we detected high (micromolar) concentrations of H2S in RA synovial fluid and levels correlated with clinical scores of inflammation and disease activity. What might be the consequences of the inflammatory generation of reactive species? Effects on inflammatory cell-signalling pathways certainly appear to be crucial, but in the current review we highlight the concept that ROS/RNS-mediated protein damage creates neoepitopes, resulting in autoantibody formation against proteins, e.g. type-II collagen and the complement component, C1q. These autoantibodies have been detected in inflammatory autoimmune diseases.     

Green tea extract protects against nonalcoholic steatohepatitis in ob/ob mice by decreasing oxidative and nitrative stress responses induced by proinflammatory enzymes

Oxidative and nitrative stress responses resulting from inflammation exacerbate liver injury associated with nonalcoholic steatohepatitis (NASH) by inducing lipid peroxidation and protein nitration. The objective of this study was to investigate whether the anti-inflammatory properties of green tea extract (GTE) would protect against NASH by suppressing oxidative and nitrative damage mediated by proinflammatory enzymes. Obese mice (ob/ob) and their 5-week-old C57BL6 lean littermates were fed 0%, 0.5% or 1% GTE for 6 weeks (n=12-13 mice/group). In obese mice, hepatic lipid accumulation, inflammatory infiltrates and serum alanine aminotransferase activity were markedly increased, whereas these markers of hepatic steatosis, inflammation and injury were significantly reduced among obese mice fed GTE. GTE also normalized hepatic 4-hydroxynonenal and 3-nitro-tyrosine (N-Tyr) concentrations to those observed in lean controls. These oxidative and nitrative damage markers were correlated with alanine aminotransferase (P<.05; r=0.410-0.471). Improvements in oxidative and nitrative damage by GTE were also associated with lower hepatic nicotinamide adenine dinucleotide phosphate oxidase activity. Likewise, GTE reduced protein expression levels of hepatic myeloperoxidase and inducible nitric oxide synthase and decreased the concentrations of nitric oxide metabolites. Correlative relationships between nicotinamide adenine dinucleotide phosphate oxidase and hepatic 4-hydroxynonenal (r=0.364) as well as nitric oxide metabolites and N-Tyr (r=0.598) suggest that GTE mitigates lipid peroxidation and protein nitration by suppressing the generation of reactive oxygen and nitrogen species. Further study is warranted to determine whether GTE can be recommended as an effective dietary strategy to reduce the risk of obesity-triggered NASH.     

Actions of melatonin in the reduction of oxidative stress

Melatonin was discovered to be a direct free radical scavenger less than 10 years ago. Besides its ability to directly neutralize a number of free radicals and reactive oxygen and nitrogen species, it stimulates several antioxidative enzymes which increase its efficiency as an antioxidant. In terms of direct free radical scavenging, melatonin interacts with the highly toxic hydroxyl radical with a rate constant equivalent to that of other highly efficient hydroxyl radical scavengers. Additionally, melatonin reportedly neutralizes hydrogen peroxide, singlet oxygen, peroxynitrite anion, nitric oxide and hypochlorous acid. The following antioxidative enzymes are also stimulated by melatonin: superoxide dismutase, glutathione peroxidase and glutathione reductase. Melatonin has been widely used as a protective agent against a wide variety of processes and agents that damage tissues via free radical mechanisms.     

DNA damage and reactive nitrogen species are barriers to Vibrio cholerae colonization of the infant mouse intestine

Ingested Vibrio cholerae pass through the stomach and colonize the small intestines of its host. Here, we show that V. cholerae requires at least two types of DNA repair systems to efficiently compete for colonization of the infant mouse intestine. These results show that V. cholerae experiences increased DNA damage in the murine gastrointestinal tract. Agreeing with this, we show that passage through the murine gut increases the mutation frequency of V. cholerae compared to liquid culture passage. Our genetic analysis identifies known and novel defense enzymes required for detoxifying reactive nitrogen species (but not reactive oxygen species) that are also required for V. cholerae to efficiently colonize the infant mouse intestine, pointing to reactive nitrogen species as the potential cause of DNA damage. We demonstrate that potential reactive nitrogen species deleterious for V. cholerae are not generated by host inducible nitric oxide synthase (iNOS) activity and instead may be derived from acidified nitrite in the stomach. Agreeing with this hypothesis, we show that strains deficient in DNA repair or reactive nitrogen species defense that are defective in intestinal colonization have decreased growth or increased mutation frequency in acidified nitrite containing media. Moreover, we demonstrate that neutralizing stomach acid rescues the colonization defect of the DNA repair and reactive nitrogen species defense defective mutants suggesting a common defense pathway for these mutants.     

Formation of 8-nitroguanosine in cellular RNA as a biomarker of exposure to reactive nitrogen species

Reactive nitrogen species, such as peroxynitrite, nitrogen oxides and nitryl chloride, have been implicated as a cause of diverse pathophysiological conditions, including inflammation, neurodegenerative and cardiovascular diseases and cancer. We previously reported that 8-nitroguanine is formed by reactions of guanine or calf-thymus DNA with peroxynitrite in vitro. In the present study, we have studied the formation of 8-nitroguanosine and 8-oxo-7,8-dihydroguanosine in reactions of calf-liver RNA with various reactive nitrogen species. 8-Nitroguanosine in RNA was found to be much more stable than 8-nitro-2' -deoxyguanosine in DNA, which rapidly depurinates to release 8-nitroguanine. Both 8-nitroguanosine and 8-oxo-7,8-dihydroguanosine were formed in calf-liver RNA following exposure to various reactive nitrogen species, such as synthetic peroxynitrite. They were also formed in RNA by reactive species formed from nitric oxide and superoxide anion generated concomitantly from 3-morpholino-sydnonimine (SIN-1) and those formed with myeloperoxidase or horseradish peroxidase in the presence of nitrite and hydrogen peroxide. 8-Nitroguanosine was detected by HPLC with an electrochemical detector in enzymatic hydrolyzates of RNA isolated from human lung carcinoma cells incubated with synthetic peroxynitrite. Our results indicate that 8-nitroguanosine in cellular RNA could be measured as a marker of damage caused by endogenous reactive nitrogen species in tissues and cells.     

Identification of TLR inducing Th1-responsive Leishmania donovani amastigote-specific antigens

Leishmania is known to elicit Th2 response that causes leishmaniasis progression; on the other hand, Th1 cytokines restricts amastigote growth and disease progression. In this study, we report the potential of two leishmanial antigens (65 and 98?kDa, in combination) which enhance strong macrophage effector functions, viz., production of respiratory burst enzymes, nitric oxide, and Th1 cytokines. The identification of antigens were done by resolving the crude soluble antigens on SDS-PAGE and eluted by reverse staining method. Further, RAW264.7 macrophages were challenged with eluted antigens, and the innate immune response was observed by detecting respiratory burst enzymes, nitric oxide (NOx), TNF-α, IFN-γ, IL-12, toll-like receptors (TLRs) gene expression, and TLR-signaling proteins. These antigens increased the production of nicotinamide adenine dinucleotide phosphate (NADPH) oxidase, superoxide dismutase, NOx, TNF-α, IFN-γ, IL-12, TLR2, and p38 mitogen-activated protein kinase. These antigens also induced human peripheral blood mononuclear cells proliferation and Th1 cytokine production. This study concludes that these antigens induce innate immune response as well as have prophylactic efficacy.     

A Detoxifying Oxygen Reductase in the Anaerobic Protozoan Entamoeba histolytica

We report the characterization of a bacterial-type oxygen reductase abundant in the cytoplasm of the anaerobic protozoan parasite Entamoeba histolytica. Upon host infection, E. histolytica is confronted with various oxygen tensions in the host intestine, as well as increased reactive oxygen and nitrogen species at the site of local tissue inflammation. Resistance to oxygen-derived stress thus plays an important role in the pathogenic potential of E. histolytica. The genome of E. histolytica has four genes that encode flavodiiron proteins, which are bacterial-type oxygen or nitric oxide reductases and were likely acquired by lateral gene transfer from prokaryotes. The EhFdp1 gene has higher expression in virulent than in nonvirulent Entamoeba strains and species, hinting that the response to oxidative stress may be one correlate of virulence potential. We demonstrate that EhFdp1 is abundantly expressed in the cytoplasm of E. histolytica and that the protein levels are markedly increased (up to ～5-fold) upon oxygen exposure. Additionally, we produced fully functional recombinant EhFdp1 and demonstrated that this enzyme is a specific and robust oxygen reductase but has poor nitric oxide reductase activity. This observation represents a new mechanism of oxygen resistance in the anaerobic protozoan pathogen E. histolytica.     

Inflammation, gene mutation and photoimmunosuppression in response to UVR-induced oxidative damage contributes to photocarcinogenesis

Ultraviolet (UV) radiation causes inflammation, gene mutation and immunosuppression in the skin. These biological changes are responsible for photocarcinogenesis. UV radiation in sunlight is divided into two wavebands, UVB and UVA, both of which contribute to these biological changes, and therefore probably to skin cancer in humans and animal models. Oxidative damage caused by UV contributes to inflammation, gene mutation and immunosuppression. This article reviews evidence for the hypothesis that UV oxidative damage to these processes contributes to photocarcinogenesis. UVA makes a larger impact on oxidative stress in the skin than UVB by inducing reactive oxygen and nitrogen species which damage DNA, protein and lipids and which also lead to NAD+ depletion, and therefore energy loss from the cell. Lipid peroxidation induces prostaglandin production that in association with UV-induced nitric oxide production causes inflammation. Inflammation drives benign human solar keratosis (SK) to undergo malignant conversion into squamous cell carcinoma (SCC) probably because the inflammatory cells produce reactive oxygen species, thus increasing oxidative damage to DNA and the immune system. Reactive oxygen or nitrogen appears to cause the increase in mutational burden as SK progress into SCC in humans. UVA is particularly important in causing immunosuppression in both humans and mice, and UV lipid peroxidation induced prostaglandin production and UV activation of nitric oxide synthase is important mediators of this event. Other immunosuppressive events are likely to be initiated by UV oxidative stress. Antioxidants have also been shown to reduce photocarcinogenesis. While most of this evidence comes from studies in mice, there is supporting evidence in humans that UV-induced oxidative damage contributes to inflammation, gene mutation and immunosuppression. Available evidence implicates oxidative damage as an important contributor to sunlight-induced carcinogenesis in humans.