**3.1.1** *H.pylori* **gastritis and eradication treatment**

*H. pylori* infection, which is the major cause of atrophic gastritis, is a high risk factor for gastric carcinoma (Peek & Blaser, 2002). Lipopolysacharide (LPS), a component of Gramnegative bacteria such as *H. pylori*, is a TLR4 ligand that induces inflammatory responses via NF-B expression (Maeda S. et al., 2001). NF-B, which is involved in the regulation of iNOS, had been reported to function as a tumor promoter in inflammation-associated cancer (Surh et al., 2001; Pikarsky et al., 2004). In patients with *H.pyroli*-induced gastritis or gastric ulcers, iNOS is expressed in the infiltrating inflammatory cells (Mannick et al., 1996). ROS and RNS generated by inflammatory cells may contribute to carcinogenesis through the formation of DNA base lesions, such as 8-oxodG, which can lead to a G:C-to-T:A transversion (Shibutani et al., 1991; Kawanishi & Murata, 2006). 8-oxodG, a marker of oxidative DNA damage, is found at a significantly increased level in the gastric epithelium of *H. pylori*-infected patients (Baik et al., 1996; Pignatelli et al., 2001). Therefore, in addition to 8-oxodG formation, the accumulation of 8-nitroguanine may play a key role in the initiation and/or promotion of inflammation-mediated carcinogenesis. To evaluate whether nitrative DNA damage plays a role early in the carcinogenic process triggered by *H. pylori* and the affection of *H. pylori* eradication treatment on 8-nitroguanine formation, we used a double-immunofluorescence staining procedure to compare the formation of both 8 nitroguanine and 8-oxodG in the gastric epithelium of gastritis patients with and without *H. pylori* infection, and before and after *H. pylori* eradication treatment patients.

The formation of 8-nitroguanine and 8-oxodG in gastric epithelium in gastritis patients with *H. pylori* infection is shown in Fig. 3. Notably, intense immunoreactivity of both lesions was observed to co-localize in gastric gland epithelial cells in patients with *H. pylori* infection (Fig. 3, HP(+)). On the other hand, in gastritis patients without *H. pylori* infection, little or no immunoreactivity was observed in gastric gland epithelial cells (Fig. 3, HP(-)). 8- Nitroguanine formation was observed in both the nuclei and the cytoplasm of the labeled epithelial cells, suggesting that it can form in both DNA and RNA. The 8-oxodG immunoreactivity was coincident with that of 8-nitroguanine within the nuclei of gastric gland cells and surface epithelial cells in *H. pylori*-infected patients (Fig. 3, merged labeling in yellow). Regardless of the *H. pylori* infection status, immunoreactivity of 8-nitroguanine and 8-oxodG was observed in inflammatory cells.

Moreover, we found that 8-nitroguanine formation in patients with *H. pylori* infection was dramatically decreased by eradication. After eradication, reduction of 8-nitroguanine and 8 oxodG formation in the epithelia was observed in 58% and 53% patients, respectively (Fig.

infection (Ma et al., 2004), hepatocytes of patients with chronic hepatitis C (Horiike et al., 2005), oral precancerous lesions oral lichen planus (OLP) (Chaiyarit et al., 2005) and oral leukoplakia (Ma et al., 2006), soft tissue sarcoma (Hoki et al., 2007a; Hoki et al., 2007b) and Epstein-Barr virus (EBV)-associated nasopharyngeal carcinoma (NPC) (Ma et al., 2008).

In animal models, 8-nitroguanine was formed in a mouse model of inflammatory bowel disease (IBD) (Ding et al., 2005). 8-Nitroguanine was formed in the bile duct epithelium of the liver of hamsters infected with the liver fluke, *Opisthorchis viverrini*, which causes cancer of intrahepatic bile duct (Pinlaor et al., 2003; Pinlaor et al., 2004a). The treatment with praziquantel, an antiparasitic drug, reduced 8-nitroguanine formation (Pinlaor et al., 2006).

*H. pylori* infection, which is the major cause of atrophic gastritis, is a high risk factor for gastric carcinoma (Peek & Blaser, 2002). Lipopolysacharide (LPS), a component of Gramnegative bacteria such as *H. pylori*, is a TLR4 ligand that induces inflammatory responses via NF-B expression (Maeda S. et al., 2001). NF-B, which is involved in the regulation of iNOS, had been reported to function as a tumor promoter in inflammation-associated cancer (Surh et al., 2001; Pikarsky et al., 2004). In patients with *H.pyroli*-induced gastritis or gastric ulcers, iNOS is expressed in the infiltrating inflammatory cells (Mannick et al., 1996). ROS and RNS generated by inflammatory cells may contribute to carcinogenesis through the formation of DNA base lesions, such as 8-oxodG, which can lead to a G:C-to-T:A transversion (Shibutani et al., 1991; Kawanishi & Murata, 2006). 8-oxodG, a marker of oxidative DNA damage, is found at a significantly increased level in the gastric epithelium of *H. pylori*-infected patients (Baik et al., 1996; Pignatelli et al., 2001). Therefore, in addition to 8-oxodG formation, the accumulation of 8-nitroguanine may play a key role in the initiation and/or promotion of inflammation-mediated carcinogenesis. To evaluate whether nitrative DNA damage plays a role early in the carcinogenic process triggered by *H. pylori* and the affection of *H. pylori* eradication treatment on 8-nitroguanine formation, we used a double-immunofluorescence staining procedure to compare the formation of both 8 nitroguanine and 8-oxodG in the gastric epithelium of gastritis patients with and without *H.* 

*pylori* infection, and before and after *H. pylori* eradication treatment patients.

The formation of 8-nitroguanine and 8-oxodG in gastric epithelium in gastritis patients with *H. pylori* infection is shown in Fig. 3. Notably, intense immunoreactivity of both lesions was observed to co-localize in gastric gland epithelial cells in patients with *H. pylori* infection (Fig. 3, HP(+)). On the other hand, in gastritis patients without *H. pylori* infection, little or no immunoreactivity was observed in gastric gland epithelial cells (Fig. 3, HP(-)). 8- Nitroguanine formation was observed in both the nuclei and the cytoplasm of the labeled epithelial cells, suggesting that it can form in both DNA and RNA. The 8-oxodG immunoreactivity was coincident with that of 8-nitroguanine within the nuclei of gastric gland cells and surface epithelial cells in *H. pylori*-infected patients (Fig. 3, merged labeling in yellow). Regardless of the *H. pylori* infection status, immunoreactivity of 8-nitroguanine

Moreover, we found that 8-nitroguanine formation in patients with *H. pylori* infection was dramatically decreased by eradication. After eradication, reduction of 8-nitroguanine and 8 oxodG formation in the epithelia was observed in 58% and 53% patients, respectively (Fig.

**3.1.1** *H.pylori* **gastritis and eradication treatment** 

and 8-oxodG was observed in inflammatory cells.

4A, B). Despite the complete eradication of *H. pylori*, reduced 8-nitroguanine and 8-oxodG production by infiltrating inflammatory cells were found only in 26% and 36% patients, respectively. Mean 8-nitroguanine and 8-oxodG immunoreactivities in inflammatory cells was not significantly decreased after the eradication treatment.

Fig. 3. 8-Nitroguanine and 8-oxodG formation in gastritis patients with and without *H. pylori* infection. Double immunofluorescence staining of paraffin sections shows the localization of 8-oxodG and 8-nitroguanine in the gastric epithelium. In *H. pylori*-infected patients (HP(+)), the immunoreactivity of 8-nitroguanine and 8-oxodG co-localizes primarily in the nuclei of gastric gland epithelial cells and in some inflammatory cells in the corpus (Merged). In chronic gastritis patients without *H. pylori*-infection (HP(-)), the immunoreactivity of 8-nitroguanine and 8-oxodG is observed mainly in the inflammatory cells, while the gastric gland epithelial cells displayed little or no immunoreactivity. Scale bar represents 50 m.

8-Nitroguanine, a Potential Biomarker to

al., 2004).

Evaluate the Risk of Inflammation-Related Carcinogenesis 211

The mechanisms by which *H. pylori* infection causes gastric cancer have been investigated. LPS, a component of Gram-negative bacteria, including *H. pylori*, is a ligand of TLR4. TLR4 is involved in activation of the transcription factor NF-B (Maeda H. & Akaike, 1998), which mediates expression of iNOS and various inflammatory cytokines. Alternatively, the Cagpositive *H.pylori* strain induces an intense inflammatory response, including interleukin-8 (IL-8) production by epithelial cells and subsequent production of tumor necrosis factor (TNF-) by inflammatory cells (Peek & Blaser, 2002). The host immune response to *H. pylori*  mediated by cytokines, resulting in iNOS expression, may lead to an increase in the

More than 300,000 new cases are being diagnosed with oral squamous cell carcinoma annually in the world (Sudbo & Reith, 2005). Oral leukoplakia is a precancerous lesion characterized by white plaque and hyperkeratosis (Neville & Day, 2002; Reibel, 2003). Five to 15% of leukoplakia is histologically classified as dysplasia (Suarez et al., 1998; Sudbo & Reith, 2005). The presence of epithelial dysplasia may be important in predicting malignant development. A substantial part of dysplasia is reported to develop into oral carcinoma (Lumerman et al., 1995; Sudbo & Reith, 2005). Oral lichen planus (OLP) is a chronic inflammatory mucosal disease (Scully et al., 1998). Several pathological features indicate that OLP is an immunologically mediated inflammatory response, including an intense, band-like infiltrate of predominantly T-lymphocytes subjacent to epithelium. Basal epithelial cells are the target for immune destruction by cytotoxic T-lymphocytes (Tyldesley & Appleton, 1973; Dekker et al., 1997). The most important complication of OLP is development of oral squamous cell carcinoma (OSCC) (Rajentheran et al., 1999; Mignogna et

Antioxidants have induced regression of precancerous oral lesions including leukoplakia (Enwonwu & Meeks, 1995), suggesting that ROS are involved in the pathogenesis of precancerous lesions. ROS are considered to participate in carcinogenesis by forming oxidative DNA lesions, such as 8-oxodG (Kawanishi et al., 2001; Kawanishi et al., 2002).

A chronic inflammatory infiltration was generally present in oral tissues of leukoplakia patients (Rodriguez-Perez & Banoczy, 1982). Expression of COX-2, an inflammatory mediator, was increased in oral mucosa with various lesions of leukoplakia, including hyperplasia and dysplasia (Renkonen et al., 2002; Altorki et al., 2004). These reports implicate that inflammation may mediate the development of oral cancer from leukoplakia. Therefore, we examined whether nitrative stress contributes to the development of oral

We demonstrated that accumulation of 8-nitroguanine and 8-oxodG was observed in oral epithelium of biopsy specimens from patients with OLP and OSCC, whereas no immunoreactivity was observed in normal oral mucosa (Chaiyarit et al., 2005). 8- Nitroguanine and 8-oxodG were also observed in oral epithelium of patients with leukoplakia (Ma et al., 2006). Co-localization of 8-nitroguanine and iNOS was found in oral epithelium of patients with OLP, OSCC and leukoplakia. Immunoreactivity of 3 nitrotyrosine, which is formed by protein tyrosine nitration and considered to be a

accumulation of 8-nitroguanine and 8-oxodG in gastric epithelium.

**3.1.2 Leukoplakia, oral lichen planus and oral cancer** 

carcinogenesis from leukoplakia through DNA damage.

Fig. 4. Detective 8-nitroguanine and 8-oxodG formation in the *H. pylori* eradication patients by double immunofluorescent method (A) and peroxidase anti-peroxidase immunohistochemical method (B). Positive immunoreactivity for 8-nitroguanine was predominantly localized in the nuclei and cytosol of foveolar cells, neutrophils and lymphocytes. The immunoreactivity of 8-nitroguanine and 8-oxodG on the nuclei of foveolar cells was greater reduced after successful *H. pylori* eradication. Conversely, reduced 8-nitroguanine and 8-oxodG formation in infiltrating inflammatory cells did not significantly decrease after the treatment. Scale bar represents 50 m.

Fig. 4. Detective 8-nitroguanine and 8-oxodG formation in the *H. pylori* eradication patients

foveolar cells was greater reduced after successful *H. pylori* eradication. Conversely, reduced

immunohistochemical method (B). Positive immunoreactivity for 8-nitroguanine was predominantly localized in the nuclei and cytosol of foveolar cells, neutrophils and lymphocytes. The immunoreactivity of 8-nitroguanine and 8-oxodG on the nuclei of

8-nitroguanine and 8-oxodG formation in infiltrating inflammatory cells did not

significantly decrease after the treatment. Scale bar represents 50 m.

by double immunofluorescent method (A) and peroxidase anti-peroxidase

The mechanisms by which *H. pylori* infection causes gastric cancer have been investigated. LPS, a component of Gram-negative bacteria, including *H. pylori*, is a ligand of TLR4. TLR4 is involved in activation of the transcription factor NF-B (Maeda H. & Akaike, 1998), which mediates expression of iNOS and various inflammatory cytokines. Alternatively, the Cagpositive *H.pylori* strain induces an intense inflammatory response, including interleukin-8 (IL-8) production by epithelial cells and subsequent production of tumor necrosis factor (TNF-) by inflammatory cells (Peek & Blaser, 2002). The host immune response to *H. pylori*  mediated by cytokines, resulting in iNOS expression, may lead to an increase in the accumulation of 8-nitroguanine and 8-oxodG in gastric epithelium.
