**8-Nitroguanine, a Potential Biomarker to Evaluate the Risk of Inflammation-Related Carcinogenesis**

Ning Ma1, Mariko Murata3, Shiho Ohnishi2, Raynoo Thanan2, Yusuke Hiraku3 and Shosuke Kawanishi2 *1Faculty of Health Science, Suzuka University of Medical Science, Suzuka, Mie, 2Faculty of Pharmaceutical Sciences, Suzuka University of Medical Science, Suzuka, Mie, 3Department of Environmental and Molecular Medicine, Mie University Graduate School of Medicine, Tsu, Mie, Japan* 

#### **1. Introduction**

200 Biomarker

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*Cancer,* Vol. 5, p. 83

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Recently, chronic inflammation induced by infection has been postulated to be an important risk factor of various cancers (Schetter et al., 2010; Aggarwal & Sung, 2011; Kamp et al., 2011; Rook & Dalgleish, 2011; Trinchieri, 2011). Many malignancies arise from areas of infection and inflammation (Balkwill & Mantovani, 2001; Coussens & Werb, 2002). Epidemiological and experimental studies have provided evidence showing that chronic infection and inflammation contribute to a substantial part of environmental carcinogenesis (Coussens & Werb, 2002; IARC, 2003). It has been estimated that chronic inflammation accounts for approximately 25 % of human cancers (Hussain S. P. & Harris, 2007). International Agency for Research on Cancer (IARC) has estimated that infectious diseases account for approximately 18 % of cancer cases worldwide (IARC, 2003). During inflammation, nitric oxide (NO) and reactive oxygen species (ROS) are generated from inflammatory cells and considered to play the key role in carcinogenes (Hofseth et al., 2003a; Hofseth et al., 2003b; Hussain S. P. et al., 2003; Ohshima et al., 2003). Inducible nitric oxide synthase (iNOS) catalyzes the production of NO particularly during inflammation, leading to generation of various reactive nitrogen species (RNS), such as NOx and peroxynitrite (ONOO-). RNS generated during infection with influenza viruses can mediate the formation of 8 nitroguanine, a nitrative lesion of nucleic acids, via ONOO formation (Maeda H. & Akaike, 1998; Akaike et al., 2003). 8-Nitroguanine formed in DNA is chemically unstable, and thus can be spontaneously released, resulting in the formation of an apurinic site (Yermilov et al., 1995a). The apurinic site can form a pair with adenine during DNA synthesis, leading to G:C-to-T:A transversions (Kawanishi & Hiraku, 2006) (Fig. 1). Thus, 8-nitroguanine is a potentially mutagenic DNA lesion, which can participate in initiation and promotion in the

8-Nitroguanine, a Potential Biomarker to

**2.1 Production of anti-8-nitroguanine antibody** 

**2.2 Specificity of anti-8-nitroguanine antibody** 

Evaluate the Risk of Inflammation-Related Carcinogenesis 203

Anti-8-nitroguanine polyclonal antibody was produced by a modified method (Akaike et al., 2003). 8-Nitroguanosine was incubated with sodium metaperiodate for 20 min at room temperature and then conjugated with rabbit serum albumin (RSA) for 1 h followed by incubation with sodium borohydride for 1 h. The conjugate was dialyzed against 150 mM NaCl overnight. 8-Nitroguanine–aldehyde–RSA conjugate mixed with Freund's complete adjuvant was injected in rabbit by intracutaneous administration. After 4 weeks of the immunization, the same antigen was given and the blood was taken two weeks later. We immobilized 8-nitroguanine in a cellulofine GCL-2000m column (Seikagaku Kogyo, Tokyo,

Specificity of the purified antibody was examined by a dot immunobinding assay and absorption test (Pinlaor et al., 2004a). Purified antibody gave a strong immunostaining only on the spot of 8-nitroguanine conjugate (Fig. 2A). The immunoreactivity disappeared only when the antibody was pre-incubated with 8-nitroguanine. In contrast, immunoreactivity with 8-nitroguanine conjugate did not disappear when the antibody was preincubated with 3-nitrotyrosine, guanosine, 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodG),

Fig. 2. Dot immunobinding assay and absorption test of anti-8-nitroguanine antibody.

**2. Immunohistochemical identification of 8-nitroguanine** 

Japan), and then purified the antibody by affinity chromatography.

deoxyguanosine, 8-bromoguanosine, and xanthosine (Fig. 2B).

infection-related carcinogenesis (Loeb & Preston, 1986; Kawanishi et al., 2006). Our studies have demonstrated that 8-nitroguanine is formed at the sites of carcinogenesis in humans and experimental animals (Ma et al., 2004; Pinlaor et al., 2004b; Ding et al., 2005; Horiike et al., 2005; Ma et al., 2006; Hoki et al., 2007a; Hoki et al., 2007b; Fujita et al., 2008; Ma et al., 2008; Tanaka et al., 2008; Ma et al., 2009; Ma et al., 2010). Moreover, our studies have demonstrated that 8-nitroguanine was formed in Oct3/4-positve stem cells in *S. haematobium*-associated cystitis and cancer tissues. Inflammation by *S. haematobium* infection may increase the number of mutant stem cells, in which iNOS-dependent DNA damage occures via NF-B activation leading to tumor development (Ma et al., 2011).

We describe the procedures of these experiments including the 8-nitroguanine antibody produce method, and employed this rabbit anti-8-nitroguanine polyclonal antibody to examine the formation and localization of 8-nitroguanine in patients and animals with inflammation related carcinogenesis by the immunohistochemical method in our laboratory. These protocols provide a detailed description of methodologies successfully used to define the pattern of 8-nitroguanine expression in pathological samples. Visualization of nuclear 8 nitroguanine expression aids in assessment of potential sites of nitrative DNA damage within inflammation-related carcinogenesis. On the basis of our results, we propose that 8 nitroguanine is a promising biomarker to evaluate the potential risk of inflammationmediated carcinogenesis.

Fig. 1. Formation of 8-nitroguanine during chronic inflammation and proposed mechanism of mutation.

infection-related carcinogenesis (Loeb & Preston, 1986; Kawanishi et al., 2006). Our studies have demonstrated that 8-nitroguanine is formed at the sites of carcinogenesis in humans and experimental animals (Ma et al., 2004; Pinlaor et al., 2004b; Ding et al., 2005; Horiike et al., 2005; Ma et al., 2006; Hoki et al., 2007a; Hoki et al., 2007b; Fujita et al., 2008; Ma et al., 2008; Tanaka et al., 2008; Ma et al., 2009; Ma et al., 2010). Moreover, our studies have demonstrated that 8-nitroguanine was formed in Oct3/4-positve stem cells in *S. haematobium*-associated cystitis and cancer tissues. Inflammation by *S. haematobium* infection may increase the number of mutant stem cells, in which iNOS-dependent DNA damage

We describe the procedures of these experiments including the 8-nitroguanine antibody produce method, and employed this rabbit anti-8-nitroguanine polyclonal antibody to examine the formation and localization of 8-nitroguanine in patients and animals with inflammation related carcinogenesis by the immunohistochemical method in our laboratory. These protocols provide a detailed description of methodologies successfully used to define the pattern of 8-nitroguanine expression in pathological samples. Visualization of nuclear 8 nitroguanine expression aids in assessment of potential sites of nitrative DNA damage within inflammation-related carcinogenesis. On the basis of our results, we propose that 8 nitroguanine is a promising biomarker to evaluate the potential risk of inflammation-

Fig. 1. Formation of 8-nitroguanine during chronic inflammation and proposed mechanism

occures via NF-B activation leading to tumor development (Ma et al., 2011).

mediated carcinogenesis.

of mutation.
