Nitrative DNA Research Articles

Genotoxic effect and nitrative DNA damage in HepG2 cells exposed to aristolochic acid

Aristolochic acid (AA), extensively used as a traditional herbal medicine, was withdrawn from the market in the last century because it was found to be a potent carcinogen in humans and animals. The aim of this study was to evaluate the genotoxic effect of AA and obtain further insight into whether the nitrative DNA damage can be induced by reactive nitrogen species (RNS), including nitric oxide (NO) and its derivative peroxynitrite (ONOO −) using human hepatoma HepG2 cells. To identify the genotoxic effect, the comet assay and micronucleus test (MNT) were performed. In the comet assay, 25–200 μM of AA caused a significant increase of DNA migration in a dose-dependent manner. A significant increase of the frequency of micronuclei was found in the range between 12.5 and 50 μM in the MNT. The results showed that AA caused DNA and chromosome damages. To elucidate the nitrative DNA damage mechanism, the level of nitrite and 8-hydroxydeoxyguanosine (8-OHdG), which can be generated by ONOO −, were monitored with the 2,3-diaminonaphthalene (DAN) assay and immunoperoxidase staining, respectively. The results showed that AA causes a significant increase in the levels of NO and formation of 8-OHdG at concentrations ≥50 μM. This observation supports the assumption that AA could exert genotoxicity probably via NO and its derivatives at higher concentrations in HepG2 cells.

A6. The role of nitrative DNA damage in inflammation-associated carcinogenesis

A6. The role of nitrative DNA damage in inflammation-associated carcinogenesis

INOS‐dependent DNA damage in patients with malignant fibrous histiocytoma in relation to prognosis

Malignant fibrous histiocytoma (MFH) is one of the most common soft tissue sarcomas. MFH has been proposed to be a lesion accompanied with inflammatory responses. During chronic inflammation, reactive nitrogen and oxygen species generated from inflammatory cells are considered to participate in carcinogenesis by causing DNA damage. 8-nitroguanine is a mutagenic nitrative DNA lesion formed during chronic inflammation. We examined whether nitrative DNA damage is related to the prognosis of MFH patients. We performed immunohistochemical analyses to examine the distribution of DNA damage and the expression of inflammation-related molecules including inducible nitric oxide synthase (iNOS), nuclear factor-kappaB (NF-kappaB), and cyclooxygenase-2 (COX-2) in clinical specimens from 25 patients with MFH. We also analyzed the correlation of DNA damage or the expression of these genes with the prognosis of MFH patients. Immunohistochemical staining revealed that the formation of 8-nitroguanine and 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodG), an oxidative DNA lesion, occurred to a much greater extent in MFH tissue specimens from deceased patients than in live patients. iNOS, NF-kappaB and COX-2 were colocalized with 8-nitroguanine in MFH tissues. It is noteworthy that the statistical analysis using the Kaplan-Meier method demonstrated strong 8-nitroguanine staining to be associated with a poor prognosis. In conclusion, 8-nitroguanine appears to participate in not only the initiation and promotion of MFH, but also in the progression of MFH, and could therefore be used as a promising biomarker to evaluate the prognosis of cancer patients.

INOS‐dependent DNA damage via NF‐κB expression in hamsters infected with Opisthorchis viverrini and its suppression by the antihelminthic drug praziquantel

Inflammation-mediated DNA damage triggered by Opisthorchis viverrini (OV) infection is a major risk factor of cholangiocarcinoma (CCA). We have recently reported that nitrative and oxidative DNA damage participates in CCA development caused by repeated infection with OV [Pinlaor et al., Carcinogenesis 2004; 25:1535-42]. Therefore, to clarify the preventive effect of the antihelminthic drug praziquantel against cholangiocarcinogenesis, we assessed the effect of this drug on nitrative and oxidative DNA damage, including the formation of 8-nitroguanine and 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodG), and the expression of inducible nitric oxide synthase (iNOS) by immunohistochemistry in OV-infected hamsters. We also examined the expression of nuclear factor-kappaB (NF-kappaB), which functions as a tumor promoter in inflammation-associated cancer. Our results showed that although 1-week treatment with praziquantel did not kill parasites completely in hamsters on days 14 and 30, this drug dramatically reduced inflammatory cell infiltration. Double immunofluorescence staining showed that drug treatment almost completely diminished OV-induced 8-nitroguanine and 8-oxodG formation in bile duct epithelial cells. Quantitative analysis using an electrochemical detector coupled to HPLC revealed that 8-oxodG level in the liver of OV-infected hamsters was significantly decreased by drug treatment (p<0.05). Western blotting and immunohistochemistry revealed that the expression of NF-kappaB and iNOS in bile duct epithelium was reduced by drug treatment. The amount of nitrate plus nitrite in the liver and plasma was significantly decreased after drug treatment. It is concluded that praziquantel can exhibit a preventive effect against OV-induced cholangiocarcinoma by inhibiting iNOS-dependent DNA damage through not only elimination of parasites but also a potential antiinflammatory effect.

Oxidative and Nitrative DNA Damage as Biomarker for Carcinogenesis with Special Reference to Inflammation

Reactive oxygen and nitrogen species are known to participate in a wide variety of human diseases. Oxidative DNAdamage is involved in chemical carcinogenesis and aging. Monocyclic chemicals induce mainly oxidative DNAdamage, whereas polycyclic chemicals can induce oxidative DNA damage in addition to DNA adduct formation. Recently, chronic infection and inflammation have been recognized as important factors for carcinogenesis. Nitrative DNA damage as well as oxidative DNA damage is induced in relation to inflammationrelated carcinogenesis. The authors examined the formation of 8-nitroguanine, a nitrative DNA lesion, in humans and animals under inflammatory conditions. An immunofluorescence labeling study demonstrated that 8-nitroguanine was strongly formed in gastric gland epithelial cells in gastritis patients with H. pylori infection, in hepatocytes in patients with hepatitis C, and in oral epithelium of patients with oral lichen planus. 8-Nitroguanine was also formed in colonic epithelial cells of model mice of inflammatory bowel diseases and patients with ulcerative colitis. Interestingly, 8-nitroguanine was formed at the sites of carcinogenesis regardless of etiology. Therefore, 8-nitroguanine could be used as a potential biomarker to evaluate the risk of inflammation- related carcinogenesis.

8-Nitroguanine, a Product of Nitrative DNA Damage Caused by Reactive Nitrogen Species: Formation, Occurrence, and Implications in Inflammation and Carcinogenesis

The authors review studies on 8-nitroguanine (8-NO(2)-G) formed by reactions of guanine, guanosine, and 2 - deoxyguanosine, either free or in DNA or RNAwith reactive nitrogen species (RNS) generated from peroxynitrite, the myeloperoxidase-H(2)O(2)-nitrite system, and others. Use of antibodies against 8-NO(2)-G has revealed increased formation of 8-NO(2)-G in various pathological conditions, including RNA virus-induced pneumonia in mice, intrahepatic bile ducts of hamsters infected with the liver fluke Opisthorchis viverrini, and gastric mucosa of patients with Helicobacter pylori-induced gastritis. Immunoreactivity has been found in the cytosol as well as in the nucleus of inflammatory cells and epithelial cells in inflamed tissues, but not in normal tissues. 8- NO(2)-G in DNA is potentially mutagenic, yielding G:C to T:A transversion, possibly through its rapid depurination to form an apurinic site and/or miscoding with adenine. 8-NO(2)-G in RNA may interfere with RNA functions and metabolism. Nitrated guanine nucleosides and nucleotides in the nucleotide pool may contribute to oxidative stress via production of superoxide mediated by various reductases and may disturb or modulate directly various important enzymes such as GTP-binding proteins and cGMP-dependent enzymes. Further studies are warranted to establish the roles of 8-NO(2)-G in various pathophysiological conditions and inflammation-associated cancer.

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.

8-Nitroguanine formation in oral leukoplakia, a premalignant lesion

Oral leukoplakia is a premalignant lesion associated with development of oral cancer. To clarify the mechanism of development of oral carcinogenesis from leukoplakia, we examined DNA damage in oral epithelium of biopsy specimens of patients with leukoplakia by immunohistochemical methods. Histological changes, such as epithelial dysplasia and infiltration of inflammatory cells were observed in oral tissues of leukoplakia patients. A double immunofluorescence labeling study demonstrated that the accumulation of mutagenic 8-nitroguanine, an indicator of nitrative DNA damage, and 8-oxo-7,8-dihydro-2'-deoxyguanosine, an indicator of oxidative DNA damage, was apparently observed in the oral epithelium of patients with leukoplakia, whereas little or no immunoreactivity was observed in normal oral mucosa. Expression of inducible nitric oxide synthase (iNOS) was also observed in oral epithelium of leukoplakia patients. Immunoreactivity of 3-nitrotyrosine, an indicator of nitrative stress, was observed in oral epithelial cells and colocalized with 8-nitroguanine. Moreover, proliferating cell nuclear antigen and p53 were expressed in 8-nitroguanine-positive epithelial cells in the basal layer. These results suggest that iNOS-mediated nitrative stress contributes to development of oral carcinogenesis from leukoplakia through DNA damage as well as oxidative stress.

Nitrative and oxidative DNA damage in oral lichen planus in relation to human oral carcinogenesis

Oral lichen planus (OLP) is a chronic inflammatory disease, which has been clinically associated with development to oral cancer. A double immunofluorescence labeling study found that 8-nitroguanine and 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodG) accumulated in oral epithelium in OLP and oral squamous cell carcinoma (OSCC) biopsy specimens, whereas little or no immunoreactivity was observed in normal oral mucosa. Colocalization of 8-nitroguanine and inducible nitric oxide synthase (iNOS) was found in oral epithelium of OLP and OSCC. Immunoreactivity of 3-nitrotyrosine, which is formed by protein tyrosine nitration and is considered to be a biochemical marker for inflammation, was also observed in oral epithelial cells and colocalized with 8-nitroguanine. Accumulation of p53 was more strongly observed in oral epithelium in OSCC than OLP, whereas there was no p53 accumulation in normal oral mucosa. Our findings demonstrate that iNOS-dependent DNA damage in OLP may lead to p53 accumulation in not only OLP but also OSCC. We conclude that the formation of potentially mutagenic DNA lesions including 8-nitroguanine and 8-oxodG may contribute to the development of oral cancer from OLP.

Nitrative DNA damage in inflammation and its possible role in carcinogenesis

Chronic inflammation has long been recognized as a risk factor for human cancer at various sites. Examples include Helicobacter pylori-induced gastritis for gastric cancer, inflammatory bowel disease (ulcerative colitis and Crohn's disease) for colorectal cancer and chronic viral hepatitis for liver cancer. Here we review the role in carcinogenesis of nitrative damage to nucleic acids, DNA and RNA, which occurs during inflammation through the generation of reactive nitrogen species, such as peroxynitrite, nitroxyl, and nitrogen dioxide. Enhanced formation of 8-nitroguanine, representative of nitrative damage to nucleobases, has been detected in various inflammatory conditions. The biochemical nature of DNA damage mediated by reactive nitrogen species is discussed in relation to its possible involvement in mutations, genetic instability, and cell death. Better understanding of the mechanisms and role of such nitrative damage in chronic inflammation-associated human cancer is a necessary basis to develop new strategies for cancer prevention by modulating the process of inflammation.

Inducible nitric oxide synthase‐dependent DNA damage in mouse model of inflammatory bowel disease

Increased cancer risk occurs in inflammatory bowel disease (IBD) undergoing long-term chronic inflammation. To evaluate whether inducible nitric oxide synthase (iNOS)-dependent DNA damage plays a role in the carcinogenic process triggered by IBD, we prepared a mouse model of IBD induced by transfer of CD45RBhighCD4+ T cells lacking regulatory T cells to female severe combined immunodeficiency (SCID) mice. CD45RBhighCD4+ T cells were isolated from mouse spleen after staining with fluorescein isothiocyanate (FITC)-conjugated anti-CD45RB monoclonal antibody, followed by anti-FITC-conjugated microbeads. This IBD mouse model showed that the bodyweight increased with aging to a lesser extent than non-treated controls, and that the intestine was shortened. Pathological findings of this mouse model, which showed severe inflammation in colon tissues, were similar to IBD patients. Double immunofluorescence technique revealed that both 8-nitroguanine and 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodG) were formed mainly in epithelial cells of the IBD mouse model. 8-Nitroguanine was formed in most of 8-oxodG-immunoreactive nuclei of epithelial cells. iNOS, proliferating cell nuclear antigen and p53 protein were also expressed in the colon epithelium. These results indicate that nitrative DNA damage, as well as oxidative DNA damage, is induced in colon epithelial cells of the IBD mouse model followed by proliferation of these cells, which may contribute to colon carcinogenesis.

Nitrative and oxidative DNA damage in intrahepatic cholangiocarcinoma patients in relation to tumor invasion

Nitrative and oxidative DNA damage such as 8-nitroguanine and 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodG) formation has been implicated in initiation and/or promotion of inflammation-mediated carcinogenesis. The aim of this study is to clarify whether these DNA lesions participate in the progression of intrahepatic cholangiocarcinoma. We investigated the relation of the formation of 8-nitroguanine and 8-oxodG and the expression of hypoxia-inducible factor-1alpha (HIF-1alpha) with tumor invasion in 37 patients with intra-hepatic cholangiocarcinoma. Immunohistochemical analyses revealed that 8-nitroguanine and 8-oxodG formation occurred to a much greater extent in cancerous tissues than in non-cancerous tissues. HIF-1alpha could be detected in cancerous tissues in all patients, suggesting low oxygen tension in the tumors. HIF-1alpha expression was correlated with inducible nitric oxide synthase (iNOS) expression (r = 0.369 and P = 0.025) and 8-oxodG formation (r = 0.398 and P = 0.015). Double immunofluorescence study revealed that iNOS and HIF-1alpha co-localized in cancerous tissues. Notably, the formation of 8-oxodG was correlated significantly with lymphatic invasion (r = 0.386 and P = 0.018). Moreover, 8-nitroguanine and 8-oxodG in non-cancerous tissues were associated significantly with neural invasion (P = 0.042 and P = 0.026, respectively). These results suggest that reciprocal activation between HIF-1alpha and iNOS mediates persistent DNA damage, which induces tumor invasiveness via mutations, resulting in poor prognosis. The formation of 8-nitroguanine and 8-oxodG plays an important role in multiple steps of genetic changes leading to tumor progression, including invasiveness.

Mechanism of NO-mediated oxidative and nitrative DNA damage in hamsters infected with Opisthorchis viverrini: a model of inflammation-mediated carcinogenesis

Inflammation mediated by infection is an important factor causing carcinogenesis. Opisthorchis viverrini (OV) infection is a risk factor of cholangiocarcinoma (CHCA), probably through chronic inflammation. Formation of 8-nitroguanine and 8-oxo-7,8-dihydro-2′-deoxyguanosine (8-oxodG), and expression of inducible nitric oxide synthase (iNOS) and heme oxygenase-1 (HO-1) were assessed in the liver of hamsters infected with OV. We newly produced specific anti-8-nitroguanine antibody without cross-reaction. Double immunofluorescence staining revealed that 8-oxodG and 8-nitroguanine were formed mainly in the same inflammatory cells and epithelium of bile ducts from day 7 and showed the strongest immunoreactivity on days 21 and 30, respectively. It is noteworthy that 8-oxodG and 8-nitroguanine still remained in epithelium of bile ducts on day 180, although amount of alanine aminotransferase activity returned to normal level. A time course of 8-nitroguanine was associated with iNOS expression. Furthermore, this study demonstrated that HO-1 expression and subsequent iron accumulation may be involved in enhancement of oxidative DNA damage in epithelium of small bile ducts. In conclusion, nitrative and oxidative DNA damage via iNOS expression in hamsters infected with OV may participate in CHCA carcinogenesis.