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We have investigated the mechanisms of oxidative and nitrative DNA damage induced by various inflammatory conditions. In relation to inflammation-related carcinogenesis, we examined the formation of 8-nitroguanine and 8-oxodG in human samples and animals. It is noteworthy that DNA damage was specifically induced at sites of carcinogenesis under various inflammatory conditions. In human samples, 8-nitroguanine formation was observed in gastric gland epithelial cells of patients with *H. pylori* infection (Ma et al., 2004) and in hepatocytes of patients with chronic hepatitis C (Horiike et al., 2005). 8-Nitroguanine was also formed in oral epithelium of OLP and OSCC patients (Chaiyarit et al., 2005; Ma et al., 2006). Moreover, in hamsters infected with the liver fluke *Opisthorchis viverrini* causing cholangiocarcinoma, 8-nitroguanine formation was induced in bile duct epithelium (Pinlaor et al., 2004b). 8-Nitroguanine formation was also found in colonic gland epithelial cells of mouse model of IBD (Ding et al., 2005). Therefore, 8-nitroguanine could be used as a potential biomarker to evaluate the risk of inflammation-related carcinogenesis. Recently, 8 nitroguanosine has been reported to be a highly redox-active molecule that strongly stimulates O2•-generation from NADPH-dependent reductases (Sawa et al., 2003). 8- Nitroguaninemay be a cofactor for redox reaction and cell signaling implicated in diverse physiological and pathological events (Zaki et al., 2005). More importantly, experimental evidence has suggested that 8-nitroguanine is a mutagenic DNA lesion, which preferentially leads to G:C-to-T:A transversions (Yermilov et al., 1995b; Suzuki et al., 2005), in addition to 8-oxodG (Shibutani et al., 1991; Bruner et al., 2000). Indeed, G:C-to-T:A transversions have been observed *in vivo* in the *ras* gene (Bos, 1988) and the *p53* tumor suppressor gene in lung and liver cancer (Takahashi et al., 1989; Prahalad et al., 1999). We also investigated the role of DNA damage in carcinogenesis initiated by *K-ras* mutation, using conditional transgenic mice. Immunohistochemical analysis revealed that mutagenic 8-nitroguanine and 8-oxodG were apparently formed in adenocarcinoma caused by mutated *K-ras*. 8-Nitroguanine was co-localized with iNOS, NF-B, IKK, MAPK, MEK, and mutated *K-ras*, suggesting that oncogenic *K-ras* causes additional DNA damage via signaling pathway involving these molecules. It is noteworthy that *K-ras* mutation mediates not only cell over-proliferation but also the accumulation of mutagenic DNA lesions, leading to carcinogenesis (Ohnishi et al., 2011). These findings imply that DNA damage mediated by ROS and RNS may participate in carcinogenesis via activation of protooncogenes and inactivation of tumor suppressor genes. In conclusion, oxidative and nitrative DNA damage could be promising biomarkers to evaluate the risk of carcinogenesis induced by a wide variety of chemicals and inflammatory conditions.

### **5. Acknowledgments and funding**

This work was supported by a Grant-in-Aid from the Ministry of Education, Culture, Sports, Science and Technology of Japan and Otsuka Pharmaceutical Co., Ltd. Japan.

#### **6. References**

Aggarwal, B. B. and Sung B. (2011). The relationship between inflammation and cancer is analogous to that between fuel and fire. *Oncology (Williston Park),* Vol.25, No.5 (Apr 30), pp. (414-418), 0890-9091 (Print)

We have investigated the mechanisms of oxidative and nitrative DNA damage induced by various inflammatory conditions. In relation to inflammation-related carcinogenesis, we examined the formation of 8-nitroguanine and 8-oxodG in human samples and animals. It is noteworthy that DNA damage was specifically induced at sites of carcinogenesis under various inflammatory conditions. In human samples, 8-nitroguanine formation was observed in gastric gland epithelial cells of patients with *H. pylori* infection (Ma et al., 2004) and in hepatocytes of patients with chronic hepatitis C (Horiike et al., 2005). 8-Nitroguanine was also formed in oral epithelium of OLP and OSCC patients (Chaiyarit et al., 2005; Ma et al., 2006). Moreover, in hamsters infected with the liver fluke *Opisthorchis viverrini* causing cholangiocarcinoma, 8-nitroguanine formation was induced in bile duct epithelium (Pinlaor et al., 2004b). 8-Nitroguanine formation was also found in colonic gland epithelial cells of mouse model of IBD (Ding et al., 2005). Therefore, 8-nitroguanine could be used as a potential biomarker to evaluate the risk of inflammation-related carcinogenesis. Recently, 8 nitroguanosine has been reported to be a highly redox-active molecule that strongly stimulates O2•-generation from NADPH-dependent reductases (Sawa et al., 2003). 8- Nitroguaninemay be a cofactor for redox reaction and cell signaling implicated in diverse physiological and pathological events (Zaki et al., 2005). More importantly, experimental evidence has suggested that 8-nitroguanine is a mutagenic DNA lesion, which preferentially leads to G:C-to-T:A transversions (Yermilov et al., 1995b; Suzuki et al., 2005), in addition to 8-oxodG (Shibutani et al., 1991; Bruner et al., 2000). Indeed, G:C-to-T:A transversions have been observed *in vivo* in the *ras* gene (Bos, 1988) and the *p53* tumor suppressor gene in lung and liver cancer (Takahashi et al., 1989; Prahalad et al., 1999). We also investigated the role of DNA damage in carcinogenesis initiated by *K-ras* mutation, using conditional transgenic mice. Immunohistochemical analysis revealed that mutagenic 8-nitroguanine and 8-oxodG were apparently formed in adenocarcinoma caused by mutated *K-ras*. 8-Nitroguanine was co-localized with iNOS, NF-B, IKK, MAPK, MEK, and mutated *K-ras*, suggesting that oncogenic *K-ras* causes additional DNA damage via signaling pathway involving these molecules. It is noteworthy that *K-ras* mutation mediates not only cell over-proliferation but also the accumulation of mutagenic DNA lesions, leading to carcinogenesis (Ohnishi et al., 2011). These findings imply that DNA damage mediated by ROS and RNS may participate in carcinogenesis via activation of protooncogenes and inactivation of tumor suppressor genes. In conclusion, oxidative and nitrative DNA damage could be promising biomarkers to evaluate the risk of carcinogenesis induced by a wide variety of chemicals and

This work was supported by a Grant-in-Aid from the Ministry of Education, Culture, Sports,

Aggarwal, B. B. and Sung B. (2011). The relationship between inflammation and cancer is

analogous to that between fuel and fire. *Oncology (Williston Park),* Vol.25, No.5 (Apr

Science and Technology of Japan and Otsuka Pharmaceutical Co., Ltd. Japan.

**4. Concluding** 

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**11** 

*2Merck KGaA, 1Lithuania 2Germany* 

**Profiling of Endogenous Peptides by** 

Egle Machtejeviene1 and Egidijus Machtejevas2

*1Lithuanian University of Health Sciences,* 

**Multidimensional Liquid Chromatography** 

The state of the organism is reflected to the key process in the living body - protein metabolism. Proteomics is the large-scale study of gene expression at the protein level, which will ultimately provide direct measurement of protein expression levels and insight into the activity state of all relevant proteins (Pandey & Mann, 2000). The proteome analysis usually includes the following strategies: native protein pre-separation, then digestion followed by separation and identification, or alternatively straight digestion, separation and identification by mass spectrometry. Therefore, starting with one protein, after digestion we will end up with approximately 30 to 70 short peptide fragments. Identification of only very few of them will provide sufficient information which protein was present in the sample. The subproject of proteomics, namely the study of all peptides expressed by a certain cell, organ or organism, is termed peptidomics. The term was introduced in 2001 (Clynen et al., 2003). Peptides often have very specific functions as mediators and indicators of biological processes. They play important roles as messengers, *e.g.*, as hormones, growth factors, and cytokines, and thus have a high impact on health and disease. Peptidomics comprises not only peptides, originally synthesized by an organism to perform a certain task, but also degradation products of proteins (degradome). Therefore, proteolytic cleavage of proteins leads to peptides as indicators of protease activity, degradation, and degeneration therefore it is also reflects the organism state. The sensitivity of proteomics and peptidomics suffers from the lack of an amplification method, analogous to the polymerase chain reaction, to reveal and quantify the presence of low-abundance proteinaceous constituents therefore the display level is difficult. These challenges motivate the researches to develop reliable analytical platforms. Shortcomings in throughput are due to the absence of technologies that can deliver fast and parallel quantitative analysis of complex peptide distributions in an automated fashion. In the future, when peptidomics will be more analyzed and understood, and biomarkers identified straight capture step of biomarkers from complex bio-sample might be used. Peptidomics especially challenges the need for robust, automated, and sensitive high-throughput technologies. Most single-dimension separations lack sufficient resolution capability to resolve complex biological matrixes. For example, in human blood serum, 90 % of the protein content of serum is composed of 10 basic proteins. The remaining 10 % of serum consists of trace amounts of millions of different proteins. Thus, partial

**1. Introduction**

suppression of NF-kappa B activation. *Mutat Res,* Vol.480-481, (Sep 1), pp. (243- 268), 0027-5107 (Print)

