Oxidative and nitrative DNA damage in animals and patients with inflammatory diseases in relation to inflammation-related carcinogenesis

Infection and chronic inflammation are proposed to contribute to carcinogenesis through inflammation-related mechanisms. Infection with hepatitis C virus, Helicobacter pylori and the liver fluke, Opisthorchis viverrini (OV), are important risk factors for hepatocellular carcinoma (HCC), gastric cancer and cholangiocarcinoma, respectively. Inflammatory bowel diseases (IBDs) and oral diseases, such as oral lichen planus (OLP) and leukoplakia, are associated with colon carcinogenesis and oral squamous cell carcinoma (OSCC), respectively. We performed a double immunofluorescence labeling study and found that nitrative and oxidative DNA lesion products, 8-nitroguanine and 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodG), were formed and inducible nitric oxide synthase (iNOS) was expressed in epithelial cells and inflammatory cells at the site of carcinogenesis in humans and animal models. Antibacterial, antiviral and antiparasitic drugs dramatically diminished the formation of these DNA lesion markers and iNOS expression. These results suggest that oxidative and nitrative DNA damage occurs at the sites of carcinogenesis, regardless of etiology. Therefore, it is considered that excessive amounts of reactive nitrogen species produced via iNOS during chronic inflammation may play a key role in carcinogenesis by causing DNA damage. On the basis of our results, we propose that 8-nitroguanine is a promising biomarker to evaluate the potential risk of inflammation-mediated carcinogenesis.     

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.     

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.     

Oxidative and nitrative DNA damage: key events in opisthorchiasis-induced carcinogenesis

Chronic inflammation induced by liver fluke (Opisthorchis viverrini) infection is the major risk factor for cholangiocarcinoma (CCA) in Northeastern Thailand. Increased levels of proinflammatory cytokines and nuclear factor kappa B that control cyclooxygenase-2 and inducible nitric oxide activities, disturb the homeostasis of oxidants/anti-oxidants and DNA repair enzymes, all of which appear to be involved in O. viverrini-associated inflammatory processes and CCA. Consequently oxidative and nitrative stress-related cellular damage occurs due to the over production of reactive oxygen and nitrogen species in inflamed target cells. This is supported by the detection of high levels of oxidized DNA and DNA bases modified by lipid peroxidation products in both animal and human tissues affected by O. viverrini-infection. Treatment of opisthorchiasis patients with praziquantel, an anti- trematode drug was shown to reduce inflammation-mediated tissue damage and carcinogenesis. The principal mechanisms that govern the effects of inflammation and immunity in liver fluke-associated cholangiocarcinogenesis are reviewed. The validity of inflammation-related biomolecules and DNA damage products to serve as predictive biomarkers for disease risk evaluation and intervention is discussed.     

Chemical basis of inflammation-induced carcinogenesis

Chronic inflammation induced by biological, chemical, and physical factors has been associated with increased risk of human cancer at various sites. Inflammation activates a variety of inflammatory cells, which induce and activate several oxidant-generating enzymes such as NADPH oxidase, inducible nitric oxide synthase, myeloperoxidase, and eosinophil peroxidase. These enzymes produce high concentrations of diverse free radicals and oxidants including superoxide anion, nitric oxide, nitroxyl, nitrogen dioxide, hydrogen peroxide, hypochlorous acid, and hypobromous acid, which react with each other to generate other more potent reactive oxygen and nitrogen species such as peroxynitrite. These species can damage DNA, RNA, lipids, and proteins by nitration, oxidation, chlorination, and bromination reactions, leading to increased mutations and altered functions of enzymes and proteins (e.g., activation of oncogene products and/or inhibition of tumor-suppressor proteins) and thus contributing to the multistage carcinogenesis process. Appropriate treatment of inflammation should be explored further for chemoprevention of human cancers.     

Surveying the damage: the challenges of developing nucleic acid biomarkers of inflammation

Epidemiological evidence points to a cause and effect relationship between chronic inflammation and human maladies such as cancer, atherosclerosis and autoimmune disease. A critical link between inflammation and disease may lie in the secretion of highly reactive oxygen and nitrogen species by macrophages and neutrophils, including hypohalous acids, nitrous anhydride, and nitrosoperoxycarbonate. Exposure of host epithelial cells to the resulting oxidation, nitration, nitrosation and halogenation chemistries leads to damage of all types of cellular molecules. Since nucleic acids sustain damage representative of the full spectrum of different chemistries and the damage likely plays a causative role in disease etiology, DNA and RNA damage products can serve as surrogates for the short-lived chemical mediators of inflammation, and as markers that provide both mechanistic understanding of the disease process and a means to quantify risk of disease. However, the very small quantities of the damaged molecules pose a challenge to the simultaneous quantification of the spectrum of lesions in the manner of proteomics or metabolomics. The goal of this Highlight is to provide an update on the chemistry of inflammation and the development of biomarkers of inflammation in the age of -omics technologies.     

Reactive Nitrogen Species Mediate DNA Damage in Helicobacter pylori-Infected Gastric Mucosa

Background:  Reactive oxygen species (ROS) and reactive nitrogen species (RNS) can play an important role in cellular injury and carcinogenesis of gastric epithelial cells infected with Helicobacter pylori . 8-OH-deoxy guanosine (8-OHdG) and 8-nitroguanine (8-NG) are markers for ROS- and RNS-mediated DNA oxidation, respectively. In this study, RNS-mediated DNA damage in gastric mucosa was observed directly using a newly developed antibody to 8-NG to clarify how H. pylori infection causes nitrative DNA damage to gastric epithelial cells.
Methods:  Immunohistochemistry with anti-8-OHdG and anti-8-NG antibodies was performed on gastric tissue samples from 45 patients (25 men and 20 women) with H. pylori -positive gastritis and 19 patients (11 men and 8 women) exhibiting successful H. pylori eradication. Histologic factors for gastric mucosal inflammation were graded according to the guidelines of the Updated Sydney system.
Results:  In corpus mucosa, 8-OHdG and 8-NG production were significantly associated with the degree of glandular atrophy, infiltration of chronic inflammatory cells and intestinal metaplasia in the glandular epithelial cells. Successful H. pylori eradication resulted in a significant reduction of chronic inflammatory cell infiltration and neutrophilic activity. Mean 8-OHdG production was lower after H. pylori eradication in both corpus and antral mucosa ( p  = .022 and .049, respectively). However, the reduction in 8-NG exhibited was more pronounced than the reduction of 8-OhdG ( p  = .004 and .007, respectively).
Conclusions:  Helicobacter pylori infection can induce inflammatory cells infiltration, which evokes DNA damage of gastric epithelial cells through ROS and RNS production. 8-NG might be a more sensitive biomarker than 8-OHdG for H. pylori -induced DNA damage in gastric mucosa.     

Oxidative stress-induced DNA damage by particulate air pollution

Exposure to ambient air particulate matter (PM) is associated with pulmonary and cardiovascular diseases and cancer. The mechanisms of PM-induced health effects are believed to involve inflammation and oxidative stress. The oxidative stress mediated by PM may arise from direct generation of reactive oxygen species from the surface of particles, soluble compounds such as transition metals or organic compounds, altered function of mitochondria or NADPH-oxidase, and activation of inflammatory cells capable of generating ROS and reactive nitrogen species. Resulting oxidative DNA damage may be implicated in cancer risk and may serve as marker for oxidative stress relevant for other ailments caused by particulate air pollution. There is overwhelming evidence from animal experimental models, cell culture experiments, and cell free systems that exposure to diesel exhaust and diesel exhaust particles causes oxidative DNA damage. Similarly, various preparations of ambient air PM induce oxidative DNA damage in in vitro systems, whereas in vivo studies are scarce. Studies with various model/surrogate particle preparations, such as carbon black, suggest that the surface area is the most important determinant of effect for ultrafine particles (diameter less than 100 nm), whereas chemical composition may be more important for larger particles. The knowledge concerning mechanisms of action of PM has prompted the use of markers of oxidative stress and DNA damage for human biomonitoring in relation to ambient air. By means of personal monitoring and biomarkers a few studies have attempted to characterize individual exposure, explore mechanisms and identify significant sources to size fractions of ambient air PM with respect to relevant biological effects. In these studies guanine oxidation in DNA has been correlated with exposure to PM(2.5) and ultrafine particles outdoor and indoor. Oxidative stress-induced DNA damage appears to an important mechanism of action of urban particulate air pollution. Related biomarkers and personal monitoring may be useful tools for risk characterization.     

Inflammation-induced DNA damage,mutations and cancer

The relationships between inflammation and cancer are varied and complex. An important connection linking inflammation to cancer development is DNA damage. During inflammation reactive oxygen and nitrogen species (RONS) are created to combat pathogens and to stimulate tissue repair and regeneration, but these chemicals can also damage DNA, which in turn can promote mutations that initiate and promote cancer. DNA repair pathways are essential for preventing DNA damage from causing mutations and cytotoxicity, but RONS can interfere with repair mechanisms, reducing their efficacy. Further, cellular responses to DNA damage, such as damage signaling and cytotoxicity, can promote inflammation, creating a positive feedback loop. Despite coordination of DNA repair and oxidative stress responses, there are nevertheless examples whereby inflammation has been shown to promote mutagenesis, tissue damage, and ultimately carcinogenesis. Here, we discuss the DNA damage-mediated associations between inflammation, mutagenesis and cancer.     

Oxidative and nitrative modifications of enkephalins by human neutrophils: effect of nitroenkephalin on leukocyte functional responses

Neutrophils play a major role in acute inflammation by generating reactive oxygen/nitrogen species. Opioid peptides, including enkephalins, are present at inflammation sites. Neutrophils contribute to protect against inflammatory pain by releasing opioid peptides. In this investigation, the ability of human polymorphonuclear cells to induce oxidative and nitrative modifications of Leu-enkephalin has been investigated in vitro. Activated human neutrophils mediate the oxidation of Leu-enkephalin resulting in the production of dienkephalin. In the presence of nitrite at concentrations observed during inflammatory and infectious process (10-50 μM), nitroenkephalin, a nitrated derivative of Leu-enkephalin, is additionally formed. The yield of nitroenkephalin increases with nitrite concentration and is significantly inhibited by the addition of catalase or 4-aminobenzoic acid hydrazide (ABAH), a specific inhibitor of peroxidases. These results suggest that neutrophils induce nitration of Leu-enkephalin by a mechanism that is dependent on myeloperoxidase activity and hydrogen peroxide. Oxidative/nitrative modifications of Leu-enkephalin have been also evidenced when cells were treated with the NO-donor molecule, DEANO. The nitrated enkephalin has been examined for its effect on leukocyte functional responses. The data reveal that nitroenkephalin at micromolar concentrations inhibits superoxide anion generation and degranulation of azurophilic granules of human polymorphonuclear cells. Moreover, nitroenkephalin inhibits spontaneous apoptosis of neutrophils, as evaluated by measuring caspase-3 activity. Collectively, our data indicate that the nitrated enkephalin attenuates neutrophil activation and promotes the short-term survival of these cells, suggesting a possible role of the nitrocompound in the efficiency and resolution of inflammatory processes.     

Inflammation and cancer IV. Colorectal cancer in inflammatory bowel disease: the role of inflammation

Patients with ulcerative colitis and Crohn's disease are at increased risk for developing colorectal cancer. To date, no known genetic basis has been identified to explain colorectal cancer predisposition in these inflammatory bowel diseases. Instead, it is assumed that chronic inflammation is what causes cancer. This is supported by the fact that colon cancer risk increases with longer duration of colitis, greater anatomic extent of colitis, the concomitant presence of other inflammatory manifestations such as primary sclerosing cholangitis, and the fact that certain drugs used to treat inflammation, such as 5-aminosalicylates and steroids, may prevent the development of colorectal cancer. The major carcinogenic pathways that lead to sporadic colorectal cancer, namely chromosomal instability, microsatellite instability, and hypermethylation, also occur in colitis-associated colorectal cancers. Unlike normal colonic mucosa, however, inflamed colonic mucosa demonstrates abnormalities in these molecular pathways even before any histological evidence of dysplasia or cancer. Whereas the reasons for this are unknown, oxidative stress likely plays a role. Reactive oxygen and nitrogen species produced by inflammatory cells can interact with key genes involved in carcinogenic pathways such as p53, DNA mismatch repair genes, and even DNA base excision-repair genes. Other factors such as NF-kappaB and cyclooxygenases may also contribute. Administering agents that cause colitis in healthy rodents or genetically engineered cancer-prone mice accelerates the development of colorectal cancer. Mice genetically prone to inflammatory bowel disease also develop colorectal cancer especially in the presence of bacterial colonization. These observations offer compelling support for the role of inflammation in colon carcinogenesis.     

Polyphenol acertannin prevents TRAIL-induced apoptosis in human keratinocytes by suppressing apoptosis-related protein activation

The tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) has been implicated in the inflammatory and immune responses, and apoptosis in skin diseases, such as atopic dermatitis. Dysregulated apoptosis is associated with various pathologic conditions, including inflammation and cancer in skin. Polyphenols, including flavonoids and tannins, have been shown to have anti-oxidant, anti-inflammatory and anti-tumor effects. However, the effect of acertannin on TRAIL-induced apoptosis in keratinocytes has not been determined. To assess the preventive effect of acertannin on apoptosis-mediated skin inflammation, we investigated the effect of acertannin on TRAIL-induced apoptosis in human keratinocytes. TRAIL induced nuclear damage, decreased Bid, Bcl-2, Bcl-xL and survivin protein levels, increased Bax levels, induced cytochrome c release, activated caspases (-8, -9 and -3) and increased tumor suppressor p53 levels. Acertannin prevented the TRAIL-induced formation of reactive oxygen/nitrogen species, apoptosis-related protein activation and cell death. The results suggest that acertannin may reduce apoptotic effect of TRAIL on human keratinocytes by suppressing the activation of the caspase-8- and Bid-pathways and the mitochondria-mediated apoptotic pathway, leading to caspase-3 activation. The preventive effect of acertannin on TRAIL-induced apoptosis may be associated with the inhibitory effect on formation of reactive oxygen/nitrogen species. Acertannin may prevent the TRAIL-induced apoptosis-mediated skin inflammation.     

Interplay between DNA repair and inflammation,and the link to cancer

Abstract

DNA damage and repair are linked to cancer. DNA damage that is induced endogenously or from exogenous sources has the potential to result in mutations and genomic instability if not properly repaired, eventually leading to cancer. Inflammation is also linked to cancer. Reactive oxygen and nitrogen species (RONs) produced by inflammatory cells at sites of infection can induce DNA damage. RONs can also amplify inflammatory responses, leading to increased DNA damage. Here, we focus on the links between DNA damage, repair, and inflammation, as they relate to cancer. We examine the interplay between chronic inflammation, DNA damage and repair and review recent findings in this rapidly emerging field, including the links between DNA damage and the innate immune system, and the roles of inflammation in altering the microbiome, which subsequently leads to the induction of DNA damage in the colon. Mouse models of defective DNA repair and inflammatory control are extensively reviewed, including treatment of mouse models with pathogens, which leads to DNA damage. The roles of microRNAs in regulating inflammation and DNA repair are discussed. Importantly, DNA repair and inflammation are linked in many important ways, and in some cases balance each other to maintain homeostasis. The failure to repair DNA damage or to control inflammatory responses has the potential to lead to cancer.     

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.     

DNA damage and autophagy

Both exogenous and endogenous agents are a threat to DNA integrity. Exogenous environmental agents such as ultraviolet (UV) and ionizing radiation, genotoxic chemicals and endogenous byproducts of metabolism including reactive oxygen species can cause alterations in DNA structure (DNA damage). Unrepaired DNA damage has been linked to a variety of human disorders including cancer and neurodegenerative disease. Thus, efficient mechanisms to detect DNA lesions, signal their presence and promote their repair have been evolved in cells. If DNA is effectively repaired, DNA damage response is inactivated and normal cell functioning resumes. In contrast, when DNA lesions cannot be removed, chronic DNA damage triggers specific cell responses such as cell death and senescence. Recently, DNA damage has been shown to induce autophagy, a cellular catabolic process that maintains a balance between synthesis, degradation, and recycling of cellular components. But the exact mechanisms by which DNA damage triggers autophagy are unclear. More importantly, the role of autophagy in the DNA damage response and cellular fate is unknown. In this review we analyze evidence that supports a role for autophagy as an integral part of the DNA damage response.     

Signaling pathways bridging microbial-triggered inflammation and cancer

Microbial-triggered inflammation protects against pathogens and yet can paradoxically cause considerable secondary damage to host tissues that can result in tissue fibrosis and carcinogenesis, if persistent. In addition to classical pathogens, gut microbiota bacteria, i.e. a group of mutualistic microorganisms permanently inhabiting the gastrointestinal tract and which plays a key role in digestion, immunity, and cancer prevention, can induce inflammation-associated cancer following the alterations of their microenvironment. Emerging experimental evidence indicates that microbiota members like Escherichia coli and several other genotoxic and mutagenic pathogens can cause DNA damage in various cell types. In addition, the inflammatory response induced by chronic infections with pathogens like the microbiota members Helicobacter spp., which have been associated with liver, colorectal, cervical cancers and lymphoma, for instance, can also trigger carcinogenic processes. A microenvironment including active immune cells releasing high amounts of inflammatory signaling molecules can favor the carcinogenic transformation of host cells. Pivotal molecules released during immune response such as the macrophage migration inhibitory factor (MMIF) and the reactive oxygen and nitrogen species' products superoxide and peroxynitrite, can further damage DNA and cause the accumulation of oncogenic mutations, whereas pro-inflammatory cytokines, adhesion molecules, and growth factors may create a microenvironment promoting neoplastic cell survival and proliferation. Recent findings on the implication of inflammatory signaling pathways in microbial-triggered carcinogenesis as well as the possible role of microbiota modulation in cancer prevention are herein summarized and discussed.     

Reactive oxygen species and gastric carcinogenesis: The complex interaction between Helicobacter pylori and host

Helicobacter pylori (H. pylori) is a highly successful human pathogen that colonizes stomach in around 50% of the global population. The colonization of bacterium induces an inflammatory response and a substantial rise in the production of reactive oxygen species (ROS) and reactive nitrogen species (RNS), mostly derived from host neutrophils and gastric epithelial cells, which play a crucial role in combating bacterial infections. However, H. pylori has developed various strategies to quench the deleterious effects of ROS, including the production of antioxidant enzymes, antioxidant proteins as well as blocking the generation of oxidants. The host's inability to eliminate H. pylori infection results in persistent ROS production. Notably, excessive ROS can disrupt the intracellular signal transduction and biological processes of the host, incurring chronic inflammation and cellular damage, such as DNA damage, lipid peroxidation, and protein oxidation. Markedly, the sustained inflammatory response and oxidative stress during H. pylori infection are major risk factor for gastric carcinogenesis. In this context, we summarize the literature on H. pylori infection-induced ROS production, the strategies used by H. pylori to counteract the host response, and subsequent host damage and gastric carcinogenesis.     

Oxidative and nitrative stress markers in glaucoma

Glaucoma is a progressive optic neuropathy and is the leading cause of blindness in the United States and other industrialized countries. Elevated pressure in the eye is a risk factor for glaucoma and indeed experimental studies of induced pressure elevation in nonhuman primate's results in typical glaucomatous optic nerve damage. However, normal intraocular pressure can also lead to loss of vision in glaucoma. Although the initiating causes leading to glaucoma are unknown, oxidative and nitrative stress appears to play a role in the progressive neuronal death that is characteristic of glaucomatous optic nerve damage. Increased markers of oxidative stress that have been reported in glaucoma include protein nitrotyrosine, carbonyls in proteins, lipid oxidation products and oxidized DNA bases. Studies have also highlighted the role of nitric oxide in glaucoma by reporting the presence of inducible nitric oxide synthase in the iris-ciliary body, retina and in the glaucomatous optic nerve head of experimental rat models. This review discusses the role of reactive oxygen and nitrogen species in the pathogenesis of glaucoma and examines the relevance of antioxidants in neurodegeneration associated with the disease. It is concluded that oxidative and nitrative stress have a pathogenic role in glaucoma.