chronic inflammation induced by biological , chemical , and physical factors has been found to be associated with the increased risk of cancer in various organs [ 13 ] ( table 1 ) . inflammation activates a variety of inflammatory cells , which trigger oxidant - generating enzymes such as inducible nitric oxide synthase ( inos ) , nadph oxidase , and myeloperoxidase to produce high concentrations of free radicals including reactive nitrogen species ( rns ) and reactive oxygen species ( ros ) . overproduction of rns and ros can change the balance of oxidants and antioxidants and cause nitrative and oxidative stress which contributes to the damage of biomolecules such as dna , rna , lipid and proteins , leading to an increase in mutations , genomic instability , epigenetic changes , and protein dysfunction and play roles in the multistage carcinogenic process . ros generate 8-oxo-7,8-dihydro-2-deoxyguanosine ( 8-oxodg , also known as 8-hydroxydg ( 8-ohdg ) ) , a marker of oxidative dna damage [ 4 , 5 ] . 8-oxodg , a potentially mutagenic dna lesion , leading to the transversion of g  :  c to t  :  a ( g    t transversion ) , has been implicated in cancers triggered by infections . carcinogenic chemicals and their metabolites as well as electron transport chains in mitochondria are able to generate ros . on the other hand , nitric oxide ( no ) , a primary initiator of rns , is generated specifically during inflammation via inos in inflammatory and epithelial cells [ 5 , 8 ] . overproduction of no participates in the generation of peroxynitrite ( onoo ) , which can lead to the formation of 8-nitroguanine , an indicator of nitrative dna damage [ 9 , 10 ] . 8-nitroguanine undergoes spontaneous depurination in dna , resulting in the formation of an apurinic site . incorporated adenine can form a pair with apurinic sites during dna replication , leading to the g    t transversion   ( figure 1 ) . moreover , apurinic sites might represent major damage that requires error - prone dna polymerase  for efficient trans - lesion dna synthesis . it was reported that dna polymerase  can efficiently bypass abasic sites by extending from nucleotides inserted opposite the lesion by other dna polymerases . wu et al . suggested that cells deficient in subunits of dna polymerase  were hypersensitive to nitrative stress , and trans - lesion dna synthesis mediated by this polymerase contributes to extensive point mutations . additionally , dna polymerases  and  were also found to be involved in the incorporation of adenine opposite 8-nitroguanine during dna synthesis in a cell - free system associated with trans - lesion dna synthesis leading to the g    t transversion . therefore , 8-nitroguanine is a potential mutagenic dna lesion involved in inflammation - mediated carcinogenesis . relevantly , systematic and comprehensive genome - scale approaches by using the immunoprecipitation - based technique combined with high - density microarrays may be useful to investigate roles of dna lesions in carcinogenesis . we focus on the roles of nitrative and oxidative dna damage in infection- and inflammation - related carcinogenesis . we produced a specific anti-8-nitroguanine antibody   and examined the localization of dna lesions by immunohistochemical analysis in animal models and clinical samples ( table 1 ) . here , we review the effects of rns-/ros - mediated dna damage on genomic instability and epigenetic change in relation to carcinogenesis . liver fluke infections of opisthorchis viverrini ( o. viverrini ) are a risk factor for cholangiocarcinoma in southeast asia . o. viverrini infestations are endemic in khon kaen province , northeastern thailand , and khon kaen has the highest incidence of cholangiocarcinoma in the world . o. viverrini infections induce inflammation in both animal models   and humans . our previous studies showed that 8-oxodg and 8-nitroguanine levels were increased in o. viverrini - infected hamsters compared with uninfected control groups [ 17 , 2224 ] . in addition , dna damage was significantly increased in reinfected hamsters compared with animals infected just once . notably , repeated infection increased inos expression and 8-nitroguanine production in the epithelium of bile ducts even after a decrease in inflammatory cells . to elucidate the mechanism involved , we examined the expression of inos , nf-b , and toll - like receptor ( tlr ) 2 in mouse macrophage cell lines treated with o. viverrini crude antigens , suggesting that o. viverrini infection induced tlr2 activation with nf-b - dependent transcription and inos expression . these findings in hamsters were confirmed by the observation that 8-oxodg and 8-nitroguanine accumulated more in cancerous areas than in intrahepatic areas adjacent to tumors in surgical specimens . furthermore , an epidemiological study of o. viverrini - infected subjects and cholangiocarcinoma patients demonstrated that urinary 8-oxodg levels were significantly higher in cholangiocarcinoma patients than in o. viverrini - infected patients and healthy subjects and higher in o. viverrini - infected subjects than in healthy subjects . the urinary 8-oxodg levels in o. viverrini - infected patients significantly decreased two months after praziquantel treatment and were comparable to levels in healthy subjects one year after treatment . these results indicate that o. viverrini causes chronic and recurrent inflammation followed by the accumulation of oxidative and nitrative dna lesions , which may participate in the development of cholangiocarcinomas . helicobacter pylori is the main cause of chronic gastritis and a potential risk factor for gastric carcinoma . the molecular mechanisms behind h. pylori - induced production of ros / rns were wide ranging from activated neutrophils to h. pylori itself , as nicely reviewed by handa et al . . h. pylori infections promote the secretion of various inflammatory cytokines , contributing directly to the pronounced inflammatory response . lipopolysaccharide , a component of gram - negative bacteria such as h. pylori , is a tlr4 ligand that induces inflammatory responses via nf-b expression . nf-b , which is involved in the regulation of inos , had been reported to function as a tumor promoter in inflammation - associated cancer [ 31 , 32 ] . in patients with h. pylori - induced gastritis or gastric ulcers , the expression of inos mrna and protein was significantly increased in the epithelial cells of h. pylori - positive gastritis patients compared to h. pylori - negative patients . recently , it was also found that h. pylori in a korean isolate induced the expression of inos via ap-1 activation . our previous study   demonstrated that levels of 8-nitroguanine and 8-oxodg in gastric gland epithelium were significantly higher in gastritis patients with h. pylori infections than in those without infections . a significant accumulation of proliferating cell nuclear antigen ( pcna ) was observed in gastric gland epithelial cells in patients infected with h. pylori in comparison to those not infected . interestingly , the accumulation of pcna was closely correlated with the formation of 8-nitroguanine and 8-oxodg . collectively , the host response to h. pylori mediated nf-b expression , resulting in inos expression accompanied by 8-nitroguanine and 8-oxodg production in the gastric epithelium . 8-nitroguanine could be not only a promising biomarker for inflammation but also a useful indicator of the risk of developing gastric cancer in response to chronic h. pylori infection . cervical cancer is the second most common cancer among women worldwide and the most common cancer among women in many developing countries . inflammation is proposed to play an integral role in the development of human papilloma virus ( hpv)-induced cervical cancer . our previous study   examined the formation of 8-nitroguanine and 8-oxodg in cells of cervical intraepithelial neoplasia ( cin , grades 13 ) and condyloma acuminatum samples and compared it with the expression of the cyclin - dependent kinase inhibitor p16 , considered a biomarker for cervical neoplasia [ 3942 ] . there were no statistically significant differences in p16 expression between cin and condyloma acuminatum samples . these results suggest that high - risk hpv types promote inos - dependent dna damage , which leads to dysplastic changes and carcinogenesis . therefore , 8-nitroguanine is a more suitable and promising biomarker for evaluating the risk of inflammation - mediated cervical carcinogenesis than p16 . nasopharyngeal carcinoma ( npc ) is strongly associated with epstein - barr virus ( ebv ) infections . various transcription factors are known to participate in inos expression including signal transducers and activators of transcription ( stats ) , such as stat1 and stat3 [ 44 , 45 ] . epidermal growth factor receptor ( egfr ) physically interacts with stat3 in the nucleus , leading to transcriptional activation of inos . stat3 is repeatedly activated through phosphorylation via the expression of latent membrane protein 1 ( lmp1 ) as well as egfr [ 46 , 47 ] , and interleukin-6 ( il-6 ) is required for lmp1-mediated stat3 activation . in addition , lmp1-mediated inos expression was reported in ebv - infected epithelium cell lines , which play a role in colonization independent of anchorage and tumorigenicity in nude mice . using biopsy and surgical specimens of nasopharyngeal tissues from npc patients in southern china , we performed double immunofluorescent staining to examine the formation of 8-nitroguanine and 8-oxodg [ 49 , 50 ] . intensive immunoreactivity to inos was detected in the cytoplasm of 8-nitroguanine - positive cancer cells . dna lesions and inos expression were also observed in epithelial cells of ebv - positive patients with chronic nasopharyngitis but weaker than those in npc patients . egfr and phosphorylated stat3 were strongly expressed in cancer cells of npc patients , suggesting that the stat3-dependent mechanism is important to the carcinogenesis . il-6 was expressed mainly in inflammatory cells of nasopharyngeal tissues of ebv - infected patients . we also found that serum levels of 8-oxodg were significantly higher in npc patients than control subjects . collectively , these findings indicate that the nuclear accumulation of egfr and activation of stat3 by il-6 play a key role in inos expression and resultant dna damage , leading to ebv - related npc . hepatitis c virus ( hcv ) is a major cause of chronic hepatitis , liver cirrhosis , and hepatocellular carcinoma throughout the world . hepatocellular carcinoma arises through genetic alterations in hepatocytes during a chronic hcv infection [ 5255 ] . we investigated the extent of nucleic acid damage in hcv - infected individuals and its change after interferon treatment . immunoreactivities of 8-nitroguanine and 8-oxodg were strongly detected in the liver of patients with chronic hepatitis c , but not control subjects . 8-nitroguanine was found to be accumulated in hepatocytes particularly in the periportal area . in the sustained virological responder group after interferon therapy , the accumulation of 8-nitroguanine and 8-oxodg was markedly decreased in the liver . we observed a strong correlation between hepatic 8-oxodg staining and serum ferritin levels , suggesting the iron content to be a strong mediator of oxidative stress and iron reduction to reduce the incidence of hepatocellular carcinoma in patients with chronic hepatitis c [ 57 , 58 ] . we also demonstrated that oxidative dna damage widely occurred in the livers of patients with chronic viral hepatitis especially chronic hepatitis c , and the iron load and 8-oxodg - positive hepatocytic count was significantly higher in hcv - infected than in hbv - infected livers . it is plausible that ros production during chronic hcv infection is the result of high iron levels in hepatic tissues , which lead to progressive liver inflammation and an increased risk of developing liver cancer . these findings indicate that 8-nitroguanine and 8-oxodg are useful as biomarkers for evaluating the severity of hcv - induced chronic inflammation leading to hepatocellular carcinoma and the efficacy of chronic hepatitis c treatment . excessive and persistent production of ros / rns by inflammatory cells is considered as a hallmark of the secondary genotoxicity of nonfibrous and fibrous particles including asbestos . asbestos is a carcinogen ( iarc group1 ) causing lung cancer and malignant mesothelioma of the pleura and peritoneum . among the different types of asbestos , crocidolite ( blue asbestos ) and amosite ( brown asbestos ) inflammation is a hallmark of the response to exposure to asbestos in both animal and human models [ 68 , 69 ] . no and nitrative stress were reported to be involved in the asbestos - derived inflammatory response via myeloperoxidase , a major constituent of neutrophils which generates hypochlorous acid and rns [ 7073 ] . we performed an immunohistochemical analysis to examine the formation of 8-nitroguanine and the expression of inos and its transcription factor ( nf-b ) in the lungs of mice intratracheally administered asbestos fibers , including crocidolite and chrysolite . 8-nitroguanine was significantly detected in bronchial epithelial cells of asbestos - exposed groups compared with the untreated group . interestingly , the immunoreactivities of 8-nitroguanine , inos , and nf-b were significantly higher in the crocidolite - exposed group than in the chrysotile - exposed group . therefore , the formation of nitrative dna damage could be one of the mechanisms responsible for the difference in carcinogenic potential between crocidolite and chrysotile . ulcerative colitis and crohn 's disease , which are referred to as inflammatory bowel diseases ( ibds ) , are well known as chronic inflammatory diseases in the lower bowel . epidemiological studies have shown that the incidence of colorectal cancer in ibd patients is greater than the expected incidence in the general population . we hypothesized that an imbalance of helper and regulatory t - cell functions plays a key role in the pathogenesis of ibd . therefore , we prepared a mouse model of ibd with an imbalance of th1 and th2 and , using double immunofluorescence labeling , revealed that both 8-nitroguanine and 8-oxodg were mainly formed in epithelial cells . we observed by using clinical samples that 8-nitroguanine and 8-oxodg were formed in colon epithelium of patients with ulcerative colitis in the active stage ( figure 2 ) . of relevance , several studies have shown that inos is expressed in epithelial cells in colitis patients [ 7678 ] . in noncancerous colon tissues from patients with ulcerative colitis , inos protein levels were positively correlated with p53 serine 15 phosphorylation levels . these results suggest that nitrative dna damage , as well as oxidative dna damage , participates in colon carcinogenesis in patients with ibd . oral lichen planus ( olp ) is a chronic inflammatory mucosal disease   and a risk factor for oral squamous cell carcinoma ( oscc ) . oral leukoplakia is a precancerous lesion characterized by white plaques and hyperkeratosis [ 81 , 82 ] . we demonstrated that 8-nitroguanine and 8-oxodg accumulated in oral epithelium of biopsy specimens from patients with olp , leukoplakia , and oscc , whereas no immunoreactivity was observed in normal oral mucosa [ 62 , 83 ] . colocalization of 8-nitroguanine and inos was found in oral epithelium of patients with olp , leukoplakia , and oscc . accumulation of p53 was observed in oral epithelium in olp and leukoplakia patients , and more prominent expression of this protein was observed in oscc patients . in addition , the immunoreactivity to pcna was significantly higher in leukoplakia patients than that in normal mucosa , suggesting an increase in cell proliferation . also reported that pcna and p53 were highly expressed in oral tissues in olp patients . we conclude that inflammation - mediated dna damage and additional epithelial cell proliferation promote oral carcinogenesis . malignant fibrous histiocytoma ( mfh ) is one of the most common soft tissue sarcomas [ 85 , 86 ] and has a poor prognosis [ 87 , 88 ] . mfh has been proposed to be accompanied by inflammatory responses [ 89 , 90 ] . however , the mechanism of its inflammation - induced carcinogenesis is still unclear . we investigated dna lesions and inflammatory - related molecules including inos , nf-b , and cox-2 . immunohistochemical staining revealed that the formation of 8-nitroguanine and 8-oxodg occurred to a much greater extent in mfh tissue specimens from deceased patients than in live patients . it is worth noting that a statistical analysis using the kaplan - meier method demonstrated strong 8-nitroguanine staining to be associated with a poor prognosis . furthermore , our study demonstrated significantly higher levels of both 8-nitroguanine and hif-1 in the tissue specimens of deceased patients than in those of living subjects . survival curves analyzed by the kaplan - meier method differed significantly between the groups with high and low staining of 8-nitroguanine as well as hif-1 . these results suggest a significant role for the inos - dependent formation of 8-nitroguanine via hif-1 and nf-b in the progression of inflammation - related cancer . these results indicate that 8-nitroguanine is involved in not only the initiation of carcinogenesis but also its progression and prognosis in cases of mfh . genomic instability is a defining characteristic of most carcinogenesis through the accumulation of mutations in several tumor suppressor genes , oncogenes , and genes that are involved in maintaining genomic stability . events resulting in chromosomal instability , such as amplification and deletions of large segments of dna , reciprocal and nonreciprocal translocations , aneuploidy , and polyploidy , constitute the large - scale genomic aberrations that characterize the majority of human cancer cells and are thought to accelerate carcinogenesis [ 91 , 92 ] . degtyareva et al . demonstrated that chronic oxidative dna damage due to dna repair defects induced chromosome instability in a saccharomyces cerevisiae model . trouiller et al . showed that titanium dioxide , a risk factor for lung cancer , induced oxidative dna damage , -h2ax foci , micronuclei , and dna deletions , suggesting a link between inflammation - associated dna damage and genomic instability . the dna damage response ( ddr ) is essential for maintaining the integrity of the genome , and a failure of this response results in genomic instability and predisposition to malignancy . phosphorylated atm ( ataxia telangiectasia mutated ) plays a role in ddr to dna double - stranded breaks . impaired function of atm was reported to be involved in dna damage - induced genomic instability [ 94 , 95 ] . tnf- is a proinflammatory cytokine and also acts as an inos regulator protein [ 96 , 97 ] . reported that tnf- induced the formation of 8-oxodg and genomic instability in primary vascular endothelial cells . therefore , tnf- and a dysfunction of atm could play key roles in the integration between inos - mediated dna damage and genomic instability . recently , we observed that phosphorylated atm and -h2ax were colocalized with 8-oxodg and 8-nitroguanine in clinical samples of cholangiocarcinoma patients as shown in figure 3 , suggesting that dna base damage caused double - stranded breaks . dna lesions were found in infiltrating inflammatory cells , hepatocyte cells of nontumor areas , and cancer cells , whereas -h2ax and phosphorylated atm were expressed in only cancer cells . moreover , ddr proteins and dna lesions were detected only very weakly in normal liver tissues , suggesting that the dna double - stranded breaks were specific to cancer cells . our observations also support the idea that highly inos - dependent dna damage causes dna double - stranded breaks and genomic instability , which play important roles in inflammation - induced carcinogenesis via tnf- signaling and ddr protein dysfunction . diverse cellular functions including the regulation of inflammatory gene expression , dna repair , and cell proliferation are regulated by epigenetic changes . an important proinflammatory cytokine il-6 has been reported to control dna methylation through il-6-mediated janus kinase ( jak)/stat3 pathways [ 101105 ] . we demonstrated that il-6 modulated inos expression via stat3 and egfr in ebv - associated nasopharyngeal carcinoma . accumulating evidence makes it increasingly clear that epigenetic silencing plays an important role in ebv - associated neoplasia . we and our colleagues have found promoter hypermethylation in several candidate genes for tumor suppressor genes [ 107110 ] . histone modifications play a role in the response to dna double - stranded breaks through atm signaling to activate -h2ax , resulting in histone ubiquitination and acetylation , and destabilization and conformational changes to nucleosomes lead to dna repair . rns cause base lesions , abasic sites , and single - stranded breaks , which may be converted into double - strand breaks in cells by enzymatic processing , when the damage is in close proximity to or encountered by the replication fork . collectively , nitrative and oxidative dna damage may activate epigenetic change via il-6 signaling and the expression of ddr proteins . we investigated the formation of 8-nitroguanine and 8-oxodg at sites of carcinogenesis in various clinical specimens and animal models in relation to inflammation - related carcinogenesis . we also observed that dna lesions were formed and significantly increased in s. haematobium - induced urinary bladder cancer compared with cancer without such an infection . in addition , barrett 's esophagus , an inflammation - related disease caused by the reflux of gastric acid , also showed greater dna damage than normal esophageal tissues ( unpublished data ) . proposed roles of inflammation - related dna damage in carcinogenesis on the basis of our findings and studies in the literature [ 94 , 113 ] are summarized in figure 4 . 8-nitroguanine and 8-oxodg are formed in various inflammation - related cancers and precancerous regions in an inos - dependent manner . tnf- and il-6 are proinflammatory cytokines which play roles in the control of inos expression via the regulation of nf-b and stat3 signaling pathways . 8-nitroguanine and 8-oxodg are mutagenic lesions resulting in the g    t transversion . this type of mutation has been found to occur in vivo in the ras gene and the p53 tumor suppressor gene in various cancers . nitrative and oxidative dna damage induce not only mutations but also genomic instability and epigenetic change via tnf- and il-6 activities and dna double - stranded breaks resulting in the activation of oncogenes and inactivation of tumor suppressor genes , which may lead to inflammation - related carcinogenesis .