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[43] Kawahara, T., Teshima, S., Oka, A., Sugiyama, T., Kishi, K., Rokutan, K., & (2001, . (2001). Type I Helicobacter pylori lipopolysaccharide stimulates toll-like receptor 4 and activates mitogen oxidase 1 in gastric pit cells. Infect Immun, 69, 4382-4389.

[44] Kim, J. M., Kim, J. S., Kim, N., Kim, S. G., Jung, H. C., & Song, I. S. (2006). Compari‐ son of primary and secondary antimicrobial minimum inhibitory concentrations for Helicobacter pylori isolated from Korean patients. Int J Antimicrob Agents, 28, 6-13.

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rum. *Helicobacter*, 28-33.

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**Section 4**

**Afferent Vagal Neural Pathway in the**

**Patients**

**Development and Healing of Chronic Gastritis in**

## **Afferent Vagal Neural Pathway in the Development and Healing of Chronic Gastritis in Patients**

**Chapter 4**

**Capsaicin-Sensitive Afferentation Represents a New**

The name of capsaicin is generally used in the medical research, however this material does not contain a uniform chemical entity. Capsaicinoids covers 5 analogues and 2 homologues

It has been established that capsaicinoids interact with capsainin-sensitive afferent nerves representing a novel regulatory pathway of gastrointestinal funcions (Jancsó et al. 1967, 1968, 1970). Capsaicin-sensitive afferent nerves contain a temperature-gated ion channel called capsaicin receptor or transient receptor potencil vanilloid 1 (TRPV1) which is ex‐ pressed by a subgroup of primary afferent nociceptive neurons (Szolcsanyi, 2004). The cap‐ saicin receptor has been cloned (Caterina et al., 1997) and has been found to be linked to a cation channel. It is gated by capsaicin and other capsaicinoids (some vanilloids) by various treatments including low pH, noxius heat and various pain-producing endogenous an exog‐ enous chemicals. Thus, those sensory nerve endings possessing these ion channels are sus‐ ceptible to being stimulated in the gastric mucosa. Upon stimulation with capsaicin, these afferent fibers develop four response stages (excitation, sensory-blocking, long-term selec‐ tive neurotoxic impairment, and irreversible cell destruction), depending on the dose and duration of exposure of the component (Mózsik et al. 2001). A low dose of capsaicin (nano‐ grams to micrograms per kilogram) causes excitation of the nerve endings, and neuropepti‐ des (substance P [SP], calcitonin gene-related peptide [CGRP], and somatostatin) are released (Holzer et al. 1998, 1999) (Szolcsányi 2004). These mediators can increase mucosal blood flow by vasodilatation (Holzer et al. 1991), can activate mast cells and immunocells in

> © 2013 Czimmer et al.; licensee InTech. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

and reproduction in any medium, provided the original work is properly cited.

© 2013 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution,

**Mucosal Defensive Neural Pathway System in the**

**Gastric Mucosa in Patients with Chronic Gastritis**

Jozsef Czimmer, Imre Laszló Szabo, Janos Szolcsanyi and Gyula Mozsik

components (Mozsik et al. 2009) (Fig. 1).

http://dx.doi.org/10.5772/52883

**1. Introduction**

Additional information is available at the end of the chapter

### **Capsaicin-Sensitive Afferentation Represents a New Mucosal Defensive Neural Pathway System in the Gastric Mucosa in Patients with Chronic Gastritis**

Jozsef Czimmer, Imre Laszló Szabo, Janos Szolcsanyi and Gyula Mozsik

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/52883

#### **1. Introduction**

The name of capsaicin is generally used in the medical research, however this material does not contain a uniform chemical entity. Capsaicinoids covers 5 analogues and 2 homologues components (Mozsik et al. 2009) (Fig. 1).

It has been established that capsaicinoids interact with capsainin-sensitive afferent nerves representing a novel regulatory pathway of gastrointestinal funcions (Jancsó et al. 1967, 1968, 1970). Capsaicin-sensitive afferent nerves contain a temperature-gated ion channel called capsaicin receptor or transient receptor potencil vanilloid 1 (TRPV1) which is ex‐ pressed by a subgroup of primary afferent nociceptive neurons (Szolcsanyi, 2004). The cap‐ saicin receptor has been cloned (Caterina et al., 1997) and has been found to be linked to a cation channel. It is gated by capsaicin and other capsaicinoids (some vanilloids) by various treatments including low pH, noxius heat and various pain-producing endogenous an exog‐ enous chemicals. Thus, those sensory nerve endings possessing these ion channels are sus‐ ceptible to being stimulated in the gastric mucosa. Upon stimulation with capsaicin, these afferent fibers develop four response stages (excitation, sensory-blocking, long-term selec‐ tive neurotoxic impairment, and irreversible cell destruction), depending on the dose and duration of exposure of the component (Mózsik et al. 2001). A low dose of capsaicin (nano‐ grams to micrograms per kilogram) causes excitation of the nerve endings, and neuropepti‐ des (substance P [SP], calcitonin gene-related peptide [CGRP], and somatostatin) are released (Holzer et al. 1998, 1999) (Szolcsányi 2004). These mediators can increase mucosal blood flow by vasodilatation (Holzer et al. 1991), can activate mast cells and immunocells in

© 2013 Czimmer et al.; licensee InTech. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. © 2013 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

the mucosa (Stead 1992), and are involved in drug effects (Mózsik et al. 2004, 2005), and so‐ matostatin can elicit systemic anti-inflammatory and analgetic "sensory functions." The im‐ munodistribution of neuropeptides (SP, VIP, NPY, SOM, GAL, and TH) released from the sensory neurons and their neuroimmune function are known in *H. pylori*-positive gastritis (Sipos et al. 2006). Notwithstanding studies supported that this gastric mucosal protective mechanism provided by capsaicin-sensitive afferent nerves exists in patients with chronic gastritis, and its extent does not depend on the presence or absence of *H. pylori* infection. In other words, the gastric mucosal protection produced by capsaicin-sensitive afferent nerves is one of the general defense mechanisms against the different noxious agents that induce chronic gastritis (Dömötör et al. 2006).

and as well as in human clinical practice. The application of capsaicin in the animal experi‐ mentswas used as a specific tool to investigate those primary afferent nociceptive neurones (Szolcsanyi, 2004; Buck and Burks, 1986; Holzer, 1988; 1991; Szállasi and Blumberg, 1999) in‐

Capsaicin-Sensitive Afferentation Represents a New Mucosal Defensive Neural Pathway System in the Gastric...

http://dx.doi.org/10.5772/52883

63

Szolcsanyi and Bartho (1981) were the first authors, who clearly identified the beneficial and harmful effect of capsaicin in experimental peptic ulcer in rats, following varying doses of capsaicin. Later, Holzer undertook extensive investigations on the mode of action of capsai‐ cin on GI functions (Holzer, 1998; 1999; Buck and Burks, 1986; Szállasi and Blumberg, 1999). We also contributed during the 1980's to GI capsaicin research from studies in animal mod‐ els (Mózsik et al., 1997c). Recently the new drug, lafutidine, was introduced in the medical treatment of GI mucosal damage (Ajioka et al., 2000; 2002; Onodera et al., 1995; 1999; Takeu‐ chi, 2006). Lafutidine is a histamine histamine type 2 receptor (H2R) blocking compound

Capsaicin-sensitive vagal neural afferentation is one of the defensive mechanisms. These nerves have been shown to play a role in gastric mucosal protection by preventing drug induced mucosal injury in animals (Abdel-Salam et al. 1999; Mózsik et al. 1997a; Reinshagen at al. 1996) and by decreasing the amount of indomethacin (IND)-induced gastric micro‐ bleeding in healthy human subjects (Kang JY et al. 1995; Mózsik et al. 2004b; Mózsik et al. 2005). These mechanisms are well known to be important in protection against gastritis caused by various harmful effects. The TRVP 1 receptors were also detected in the area post‐ rema and in the nucleus tractus solitary, where the afferent fibres of the vagus nerve taper, and have key role in regulation of gastric functions i.e. secretion, motiity and protestion

The vagus nerve consists of 10% efferent nerves, 90% afferents. About 10% of these afferent nerves are capsaicin-sensitive. The amount of efferent nerves and the capsaicin-sensitive af‐ ferent nerves is roughly equal in the vagus nerve. Capsaicin exposure exerts various re‐ sponses in these afferent nerves depending on dose and exposure duration (excitation, sensory-blocking, long-term selective neurotoxic impairment and irreversible cell destruc‐ tion) (Vincze et al. 2004). During exposure to small doses of capsaicin (from ng/kg to µg/kg body weight) neurotransmitters, such as: substance P (SP), calcitonin gene-related peptide (CGRP) and somatostatin, are released from the nerve endings (Caterina et al. 1997; Mózsik et al. 2001; Holzer 1998). These mediators are responsible for increasing mucosal blood flow by vasodilatation (Holzer 1999), activation of mast cells and other immune cells in the muco‐

Capsaicin has been used in human studies, looks to be a suitable treatment tool in several fields of medicine that can be seen from several running and completes multicentric control‐ led studies worldwide. The United States Pharmacopeia (USP) describes the list of capsai‐ cins and their definition, identification, melting range, and content of capsaicin, dihydrocapsaicin and other capsaicinoids as follows (USP30-NF25. 2006 Edition, pp.1609): capsaicin contains not less than 90.0 % and more than 110.0 % of the labelled percentage of total capsaicinoids. The content of capsaicin is not less than 55 %, and the sum of the con‐ tents of capsaicin and dihydrocapsaicin should not be less than 75 %. The content of other

sa (Szolcsányi 2004; Holzer et al. 1991) and defense of gastrointestinal mucosa.

volved in the different physiological, pathological processes.

which uniquely has typical capsaicin actions on the target organ.

mechanisms.

Figure 1. and figure legend needs to be changed to the following:

**Figure legend 1: Names and chemical structures of capsaicin homologs and analogs (with knowledge and allowance of Mozsik et al. 2009). Figure 1.** Names and chemical structures of capsaicin homologs and analogs (with knowledge and allowance of Moz‐ sik et al. 2009).

**REFERENCES** - The correct year number of reference (30) is Mózsik et al. **(2004a)** - The correct year number of reference (31) is Mózsik et al. **(2004b)** - The correct year number of reference (34) is Mózsik et al. **(2009a)**  - The following reference was misorganised: The vagal nerve has a key-role in the development of gastrointestinal mucosal damage and prevention (Mozsik et al., 1982). The key-role of vagal nerve has been emphasized domi‐ nantly in the aggressive processes to gastrointestinal (GI) mucosa (such as in peptic ulcer disease, gastric mucosal damage, etc.) as evidenced from GI investigations in animal models and as well as in human clinical practice. The application of capsaicin in the animal experi‐ mentswas used as a specific tool to investigate those primary afferent nociceptive neurones (Szolcsanyi, 2004; Buck and Burks, 1986; Holzer, 1988; 1991; Szállasi and Blumberg, 1999) in‐ volved in the different physiological, pathological processes.

the mucosa (Stead 1992), and are involved in drug effects (Mózsik et al. 2004, 2005), and so‐ matostatin can elicit systemic anti-inflammatory and analgetic "sensory functions." The im‐ munodistribution of neuropeptides (SP, VIP, NPY, SOM, GAL, and TH) released from the sensory neurons and their neuroimmune function are known in *H. pylori*-positive gastritis (Sipos et al. 2006). Notwithstanding studies supported that this gastric mucosal protective mechanism provided by capsaicin-sensitive afferent nerves exists in patients with chronic gastritis, and its extent does not depend on the presence or absence of *H. pylori* infection. In other words, the gastric mucosal protection produced by capsaicin-sensitive afferent nerves is one of the general defense mechanisms against the different noxious agents that induce

Decyl vanillylamide (**33**) CH2 CH2 CH2 CH2 CH2 CH2 CH2 CH2 CH3

**Names and chemical structures of capsaicin homologs and analogs (with knowledge and**

**Figure 1.** Names and chemical structures of capsaicin homologs and analogs (with knowledge and allowance of Moz‐

The vagal nerve has a key-role in the development of gastrointestinal mucosal damage and prevention (Mozsik et al., 1982). The key-role of vagal nerve has been emphasized domi‐ nantly in the aggressive processes to gastrointestinal (GI) mucosa (such as in peptic ulcer disease, gastric mucosal damage, etc.) as evidenced from GI investigations in animal models

Nonoyl vanillylamide (**32**) CH2 CH2 CH2 CH2 CH2 CH2 CH2 CH3

Homodihydrocapsaicin II (**30**) CH2 CH2 CH2 CH2 CH2 CH2 CH CH2 CH3

Homodihydrocapsaicin I (**29**) CH3

Dihydrocapsaicin (**<sup>28</sup>** CH3 )

CH3

CH3

CH2 CH CH3 CH3

CH3

CH2 CH CH3 CH3

CH3

H C H

H C H

H C H CH CH3 CH3

CH2 CH2 CH2 CH2 CH2 CH2 CH2 CH

CH2 CH2 CH2 CH2 CH2 CH

CH2 CH2 CH2 CH2 C

O R

CH2 CH2 CH2 CH2 CH2 CH2 CH

Octanoyl vanillylamide (**31**) CH2 CH2 CH2 CH2 CH2 CH2 CH3

Nordihydrocapsaicin (**27**) CH3

Figure 1. and figure legend needs to be changed to the following:

HO

Homocapsaicin II (**26**) CH2 CH2 CH2 CH2 C

Homocapsaicin I (**25**) CH2 CH2 CH2 CH2 C

Name Structure of the "R" chain

CH3O CH2 NH C

chronic gastritis (Dömötör et al. 2006).

62 Current Topics in Gastritis - 2012

Capsaicin (**24**)

**Figure legend 1:**

**REFERENCES**

sik et al. 2009).

**allowance of Mozsik et al. 2009).**

Szolcsanyi and Bartho (1981) were the first authors, who clearly identified the beneficial and harmful effect of capsaicin in experimental peptic ulcer in rats, following varying doses of capsaicin. Later, Holzer undertook extensive investigations on the mode of action of capsai‐ cin on GI functions (Holzer, 1998; 1999; Buck and Burks, 1986; Szállasi and Blumberg, 1999). We also contributed during the 1980's to GI capsaicin research from studies in animal mod‐ els (Mózsik et al., 1997c). Recently the new drug, lafutidine, was introduced in the medical treatment of GI mucosal damage (Ajioka et al., 2000; 2002; Onodera et al., 1995; 1999; Takeu‐ chi, 2006). Lafutidine is a histamine histamine type 2 receptor (H2R) blocking compound which uniquely has typical capsaicin actions on the target organ.

Capsaicin-sensitive vagal neural afferentation is one of the defensive mechanisms. These nerves have been shown to play a role in gastric mucosal protection by preventing drug induced mucosal injury in animals (Abdel-Salam et al. 1999; Mózsik et al. 1997a; Reinshagen at al. 1996) and by decreasing the amount of indomethacin (IND)-induced gastric micro‐ bleeding in healthy human subjects (Kang JY et al. 1995; Mózsik et al. 2004b; Mózsik et al. 2005). These mechanisms are well known to be important in protection against gastritis caused by various harmful effects. The TRVP 1 receptors were also detected in the area post‐ rema and in the nucleus tractus solitary, where the afferent fibres of the vagus nerve taper, and have key role in regulation of gastric functions i.e. secretion, motiity and protestion mechanisms.

The vagus nerve consists of 10% efferent nerves, 90% afferents. About 10% of these afferent nerves are capsaicin-sensitive. The amount of efferent nerves and the capsaicin-sensitive af‐ ferent nerves is roughly equal in the vagus nerve. Capsaicin exposure exerts various re‐ sponses in these afferent nerves depending on dose and exposure duration (excitation, sensory-blocking, long-term selective neurotoxic impairment and irreversible cell destruc‐ tion) (Vincze et al. 2004). During exposure to small doses of capsaicin (from ng/kg to µg/kg body weight) neurotransmitters, such as: substance P (SP), calcitonin gene-related peptide (CGRP) and somatostatin, are released from the nerve endings (Caterina et al. 1997; Mózsik et al. 2001; Holzer 1998). These mediators are responsible for increasing mucosal blood flow by vasodilatation (Holzer 1999), activation of mast cells and other immune cells in the muco‐ sa (Szolcsányi 2004; Holzer et al. 1991) and defense of gastrointestinal mucosa.

Capsaicin has been used in human studies, looks to be a suitable treatment tool in several fields of medicine that can be seen from several running and completes multicentric control‐ led studies worldwide. The United States Pharmacopeia (USP) describes the list of capsai‐ cins and their definition, identification, melting range, and content of capsaicin, dihydrocapsaicin and other capsaicinoids as follows (USP30-NF25. 2006 Edition, pp.1609): capsaicin contains not less than 90.0 % and more than 110.0 % of the labelled percentage of total capsaicinoids. The content of capsaicin is not less than 55 %, and the sum of the con‐ tents of capsaicin and dihydrocapsaicin should not be less than 75 %. The content of other capsaicinoids should not be more than 15 %, all calculated on the dried basis. The circum‐ stances of plant cultivation, preparation, storage and other facts are summarized in the drug master file (DMF). The DMF of capsaicin originated from India was signed by Food and Drug Administration (FDA) in the United States as existing registration, which allows the usage of capsaicin as basic source for capsaicin-containing drugs. This type of capsaicinoids (briefly capsaicin) was used in our previous studies (Mozsik et al. 2009a, 2009b, 2011).

clonal antibody, the TRPV1 receptor and CGRP were labeled using polyclonal rabbit

Capsaicin-Sensitive Afferentation Represents a New Mucosal Defensive Neural Pathway System in the Gastric...

http://dx.doi.org/10.5772/52883

65

**Figure 2.** Immunohistochemical distribution of capsaicin receptor (transitoric receptor potential vanilloid) (TRPV1 in the gastric mucosa of a healthy (A) and of patient with *H. pylori* negative (B) and *H. pylori* positive (C) chronic gastritis. Arrows indicate the immunosigns in the epithelial layer of the gastric mucosa (original magnification: 100x). From the

**Figure 3.** Immunoshistochemical distribution of calcitonin gene-related peptide (CGRP) in the gastric mucosa of healthy subject (A), of patient with *H. pylori* negative (B) and *H. pylori* positive (C) chronic gastritis. Arrows demosn‐ trate the immunosign in the epithelial layer of the gastric mucosa (original magnification: 100x). From the ref. (Mózsik

ref. (Mózsik et al. 2011), with permission.

et al. 2011), with permission.

antisera (all from Alcam Ltd., UK, Cambridge) (Dömötör et al. 2006) (Figs. 2-4).

Gastritis is a pathomorphological appearance of inflammation in the gastric mucosa. Acute and chronic gastritis can be differentiated on the basis of the development and process of the disease. Chronic gastritis may be caused by different factors such as *Helicobacter pylori* infec‐ tion, bacterial overgrowt in a hypochlorhydric stomach, autoimmune mechanisms, or chem‐ ical agents such as long-term nonsteroidal anti-inflammatory drug (NSAID) treatment, and bile reflux (Owen 2003; Appelman 1994, Szabo et al. 2012), for details about mechanisms we refere to the other chapters of this book. Nowdays, the importance of *Helicobacter pylori* in‐ fection is increasing as the main causative factor in gastric diseases in humans. This bacteri‐ um is highly prevalent in many countries (Parsonnet 1995) and it increases the risk for development of gastric and duodenal ulcer disease, gastric cancer, and gastric mucosa-asso‐ ciated lymphoid tissue lymphoma (Janulaityte-Gunther et al. 2005; Mitani et al. 2004; Peng et al. 1998; Salih et al. 2005; Zhang et al. 2005a, 2005b). Dysfunction of gastrointestinal mu‐ cosal defense mechanisms is also involved in the development of gastric diseases. Capsai‐ cin-sensitive afferent nerves take part in gastric mucosal protection in animals (Mózsik et al. 1997a; Reinshagen et al 1996) and in healthy human subjects (Kang et al. 1995; Mózsik et al. 2004a, 2005a), and the presence of these fibers is proved to play a role in the development of human gastrointestinal disorders including gastritis, peptic ulcer, polyp without and with dysplasia, tumor, and inflammatory bowel diseases (Dömötör et al. 2005; Vincze et al. 2004). The immunodistribution of neuropeptides (SP, VIP, NPY, SOM, GAL, and TH) released from the sensory neurons and their neuroimmune function are known in *H. pylori*-positive gastritis (Sipos et al. 2006).

#### **2. Recent results in human studies**

Presentation of capsaicin-sensitive afferentation in the *H. pylori* positive and *H. pylori* nega‐ tive chronic gastritis in patients has been studied by Mózsik et al. (2011): The symptoms of patients sufferring from chronic gastritis with or without *H. pylori* infection (*H. pylori* posi‐ tive, n=21, age 39-68 years, screened with [14C] urea breath test, rapid urease test, Warthin-Starry silver staining and specific histological examinations; *H. pylori* negative, n=30, age 39-68 years) were nonspecific including gastric dyscomfort sensation, nausea, loss of apetite and vomiting. The gastric tissue samples from the stomach and antrum were examined by an independent histopathologist and classified of chronic gastritis according to the Sydney's System (Price et al. 1991). The immunohistological studies were carried out on formalin fixed, parafin embedded tissue samples of gastric mucosa using the peroxidase-labeled pol‐ ymer method (Lab Vision Co., Fremont, USA). SP was detected by the NC1/34HL rat mono‐ clonal antibody, the TRPV1 receptor and CGRP were labeled using polyclonal rabbit antisera (all from Alcam Ltd., UK, Cambridge) (Dömötör et al. 2006) (Figs. 2-4).

capsaicinoids should not be more than 15 %, all calculated on the dried basis. The circum‐ stances of plant cultivation, preparation, storage and other facts are summarized in the drug master file (DMF). The DMF of capsaicin originated from India was signed by Food and Drug Administration (FDA) in the United States as existing registration, which allows the usage of capsaicin as basic source for capsaicin-containing drugs. This type of capsaicinoids (briefly capsaicin) was used in our previous studies (Mozsik et al. 2009a, 2009b, 2011).

Gastritis is a pathomorphological appearance of inflammation in the gastric mucosa. Acute and chronic gastritis can be differentiated on the basis of the development and process of the disease. Chronic gastritis may be caused by different factors such as *Helicobacter pylori* infec‐ tion, bacterial overgrowt in a hypochlorhydric stomach, autoimmune mechanisms, or chem‐ ical agents such as long-term nonsteroidal anti-inflammatory drug (NSAID) treatment, and bile reflux (Owen 2003; Appelman 1994, Szabo et al. 2012), for details about mechanisms we refere to the other chapters of this book. Nowdays, the importance of *Helicobacter pylori* in‐ fection is increasing as the main causative factor in gastric diseases in humans. This bacteri‐ um is highly prevalent in many countries (Parsonnet 1995) and it increases the risk for development of gastric and duodenal ulcer disease, gastric cancer, and gastric mucosa-asso‐ ciated lymphoid tissue lymphoma (Janulaityte-Gunther et al. 2005; Mitani et al. 2004; Peng et al. 1998; Salih et al. 2005; Zhang et al. 2005a, 2005b). Dysfunction of gastrointestinal mu‐ cosal defense mechanisms is also involved in the development of gastric diseases. Capsai‐ cin-sensitive afferent nerves take part in gastric mucosal protection in animals (Mózsik et al. 1997a; Reinshagen et al 1996) and in healthy human subjects (Kang et al. 1995; Mózsik et al. 2004a, 2005a), and the presence of these fibers is proved to play a role in the development of human gastrointestinal disorders including gastritis, peptic ulcer, polyp without and with dysplasia, tumor, and inflammatory bowel diseases (Dömötör et al. 2005; Vincze et al. 2004). The immunodistribution of neuropeptides (SP, VIP, NPY, SOM, GAL, and TH) released from the sensory neurons and their neuroimmune function are known in *H. pylori*-positive

Presentation of capsaicin-sensitive afferentation in the *H. pylori* positive and *H. pylori* nega‐ tive chronic gastritis in patients has been studied by Mózsik et al. (2011): The symptoms of patients sufferring from chronic gastritis with or without *H. pylori* infection (*H. pylori* posi‐ tive, n=21, age 39-68 years, screened with [14C] urea breath test, rapid urease test, Warthin-Starry silver staining and specific histological examinations; *H. pylori* negative, n=30, age 39-68 years) were nonspecific including gastric dyscomfort sensation, nausea, loss of apetite and vomiting. The gastric tissue samples from the stomach and antrum were examined by an independent histopathologist and classified of chronic gastritis according to the Sydney's System (Price et al. 1991). The immunohistological studies were carried out on formalin fixed, parafin embedded tissue samples of gastric mucosa using the peroxidase-labeled pol‐ ymer method (Lab Vision Co., Fremont, USA). SP was detected by the NC1/34HL rat mono‐

gastritis (Sipos et al. 2006).

64 Current Topics in Gastritis - 2012

**2. Recent results in human studies**

**Figure 2.** Immunohistochemical distribution of capsaicin receptor (transitoric receptor potential vanilloid) (TRPV1 in the gastric mucosa of a healthy (A) and of patient with *H. pylori* negative (B) and *H. pylori* positive (C) chronic gastritis. Arrows indicate the immunosigns in the epithelial layer of the gastric mucosa (original magnification: 100x). From the ref. (Mózsik et al. 2011), with permission.

**Figure 3.** Immunoshistochemical distribution of calcitonin gene-related peptide (CGRP) in the gastric mucosa of healthy subject (A), of patient with *H. pylori* negative (B) and *H. pylori* positive (C) chronic gastritis. Arrows demosn‐ trate the immunosign in the epithelial layer of the gastric mucosa (original magnification: 100x). From the ref. (Mózsik et al. 2011), with permission.

TRPV1 and CGRP did not depend on the presence of *H. pylori* infection. The changes in ex‐

Capsaicin-Sensitive Afferentation Represents a New Mucosal Defensive Neural Pathway System in the Gastric...

Another surprising result was obtained in patients with *H. pylori* positive chronic gastritis before and after sucessfull eradication treatment. The extent of expression of TRPV1, CGRP remained at the same level after eradication. No significant changes were obtained in the ex‐

A B C D E

NS

\*\*\* \*\*\*

NS

NS NS NS

\*\*\*

\*\*\*

\*\*\*

http://dx.doi.org/10.5772/52883

67

\*\*\* \*\*\*

NS

NS NS

NS

A B C D E

NS

NS

<sup>A</sup> <sup>B</sup> <sup>C</sup> <sup>D</sup> <sup>E</sup> <sup>0</sup>

NS

NS

thromycin 500 mg, all two times per day, for seven days) (n=number of patients) (Mózsik et al 2012).

*n= 40 21 30 18 18*

**Figure 5.** Chamges in the expression of capsaicin receptor (TRPV1), calcitonin gene-ralated peptide (CGRP) and sub‐ stabtance P (SP) in the human gastric mucosa of healthy voluntaries(histologically intact) (A), *H. pylori* positive (B), *H. pylori* negative (C) and *H. pylori* positive before (D) and after eradication (pantoprazole 40, amoxicillin 1000 and clari‐

These findings suggest potential role of capsaicin-sensitive afferent vagal nerve in the devel‐ opment of chronic gastritis and the eradication treatment (Mózsik et al 2011): From the ex‐ perimental observations it could be concluded that actions of capsaicin are dose-dependent (Mozsik et al. 2001, 2005a, 2009b). The types of gastric mucosal injuries could be prevented by application of small doses (200 to 800µg/person) of capsaicin (Mózsik et al. 2001). Results of these observations suggest mucosal protecting effect of capsaicin (acting via the stimula‐ tion of capsaicin-sensitive afferent fibres of vagal nerve) in healthy human subjects. These observations showed that the expression of TRPV1 and CGRP increased siginicantly in infla‐

pression of SP in the gastric mucosa in these patients (Fig. 4) (Mózsik et al 2011).

\*\*\*

pression of SP were not significant between these groups of patients.

%

Expression

of TRPV1, CGRP, SP (in

percent, means

SEM)

%

%

**CGRP**

**TRPV1**

**SP**

**Figure 4.** Immonohsitochemical distribution of subastane P (SP) in gastric mucosa of healthy subject (A), of pateints with *H. pylori* negative (B) and positive (C) chronic gastritis. Arrows show the immunosigns in the epithelial layer of the gastric mucosa (original magnification: 100x). From the Ref. (Mózsik et al 2011), with permission.

Presentation of capsaicin-sensitive afferention of vagal nerve in patients with *H. pylori* pos‐ itve chronic gastritis of patients, before after after eradication treatment (Mózsik et al 2011):Very recently the same clinical and immunohistochemical examinations were carried out as those mentioned above in patients with *H. pylori* positive chronic gastritis before and after eradication treatment. These observations were carried out in 38 persons, including 20 healthy subjects and 18 patients with *H. pylori* positive gastritis. The age of persons with his‐ tologically intact gastric mucosa (controls) were between 41 and 67 years (mean=52.2 years). The age of patients (6 males, 12 females) was 39 to 68 years (mean = 56.4 years).

The time period between the first and control gastroscopy was 6 weeks. The biopsies were taken from the corpus and antrum of patients with chronic gastritis, before and after eradi‐ dation treatment, and from healthy persons. *H. pylori* positive patients underwent 7 days eradication treatment with combination of double dose PPI (pantoprazole 2x40 mg/day), amoxicillin (1000 mg twice daily) and clarithromycin (500 mg twice daily) according to cur‐ rent European guidelines (Malferteiner et al. 2007). After this one week combination treat‐ ment, patients continued to take normal dose of PPI for another week. The *H. pylori* infection was detected before and after by [14 C] urea breath test, rapid urease test, Warthing-Starry silver staining and specific histological and immmunohistological examinations. The results of eradication treatment was succesfull in 89%, the gastric histology indicated normal pic‐ ture in 22% of cases, and in 78 per cent patient the mucosa showed moderate gastritis (Lak‐ ner et al. 2011).

Expression of TRPV1 receptor, CGRP and SP in the gastric mucosa of patients with *H. pylori* negative and positive chronic gastritis, and before and after successful eradication treatment (Mózsik et al 2011): The expression of TRPV1 and CGRP increased in the gastric mucosa of patients with chronic gastritis, however, it was an unexpected that the increase expresson of TRPV1 and CGRP did not depend on the presence of *H. pylori* infection. The changes in ex‐ pression of SP were not significant between these groups of patients.

Another surprising result was obtained in patients with *H. pylori* positive chronic gastritis before and after sucessfull eradication treatment. The extent of expression of TRPV1, CGRP remained at the same level after eradication. No significant changes were obtained in the ex‐ pression of SP in the gastric mucosa in these patients (Fig. 4) (Mózsik et al 2011).

**Figure 4.** Immonohsitochemical distribution of subastane P (SP) in gastric mucosa of healthy subject (A), of pateints with *H. pylori* negative (B) and positive (C) chronic gastritis. Arrows show the immunosigns in the epithelial layer of the

Presentation of capsaicin-sensitive afferention of vagal nerve in patients with *H. pylori* pos‐ itve chronic gastritis of patients, before after after eradication treatment (Mózsik et al 2011):Very recently the same clinical and immunohistochemical examinations were carried out as those mentioned above in patients with *H. pylori* positive chronic gastritis before and after eradication treatment. These observations were carried out in 38 persons, including 20 healthy subjects and 18 patients with *H. pylori* positive gastritis. The age of persons with his‐ tologically intact gastric mucosa (controls) were between 41 and 67 years (mean=52.2 years).

The time period between the first and control gastroscopy was 6 weeks. The biopsies were taken from the corpus and antrum of patients with chronic gastritis, before and after eradi‐ dation treatment, and from healthy persons. *H. pylori* positive patients underwent 7 days eradication treatment with combination of double dose PPI (pantoprazole 2x40 mg/day), amoxicillin (1000 mg twice daily) and clarithromycin (500 mg twice daily) according to cur‐ rent European guidelines (Malferteiner et al. 2007). After this one week combination treat‐ ment, patients continued to take normal dose of PPI for another week. The *H. pylori* infection was detected before and after by [14 C] urea breath test, rapid urease test, Warthing-Starry silver staining and specific histological and immmunohistological examinations. The results of eradication treatment was succesfull in 89%, the gastric histology indicated normal pic‐ ture in 22% of cases, and in 78 per cent patient the mucosa showed moderate gastritis (Lak‐

Expression of TRPV1 receptor, CGRP and SP in the gastric mucosa of patients with *H. pylori* negative and positive chronic gastritis, and before and after successful eradication treatment (Mózsik et al 2011): The expression of TRPV1 and CGRP increased in the gastric mucosa of patients with chronic gastritis, however, it was an unexpected that the increase expresson of

gastric mucosa (original magnification: 100x). From the Ref. (Mózsik et al 2011), with permission.

The age of patients (6 males, 12 females) was 39 to 68 years (mean = 56.4 years).

ner et al. 2011).

66 Current Topics in Gastritis - 2012

**Figure 5.** Chamges in the expression of capsaicin receptor (TRPV1), calcitonin gene-ralated peptide (CGRP) and sub‐ stabtance P (SP) in the human gastric mucosa of healthy voluntaries(histologically intact) (A), *H. pylori* positive (B), *H. pylori* negative (C) and *H. pylori* positive before (D) and after eradication (pantoprazole 40, amoxicillin 1000 and clari‐ thromycin 500 mg, all two times per day, for seven days) (n=number of patients) (Mózsik et al 2012).

These findings suggest potential role of capsaicin-sensitive afferent vagal nerve in the devel‐ opment of chronic gastritis and the eradication treatment (Mózsik et al 2011): From the ex‐ perimental observations it could be concluded that actions of capsaicin are dose-dependent (Mozsik et al. 2001, 2005a, 2009b). The types of gastric mucosal injuries could be prevented by application of small doses (200 to 800µg/person) of capsaicin (Mózsik et al. 2001). Results of these observations suggest mucosal protecting effect of capsaicin (acting via the stimula‐ tion of capsaicin-sensitive afferent fibres of vagal nerve) in healthy human subjects. These observations showed that the expression of TRPV1 and CGRP increased siginicantly in infla‐

mated chronic gastritis, and no significant change was obtained in SP levels. However, these immunohistochemical results did not differ in patients with *H. pylori* positive and negative chronic gastritis. Capsaicin-sensitive afferention did not differ before and after (6 weeks) successfull eradication treatment in patients with chronic *H. pylori* positive gastritis (mean‐ while the control biopsy was normal in 22% and in 78% indicated in moderated histological picture of gastritis) (Lakner et al. 2011).

In animal experiments, different noxious agents produce the same (or basically similar) pathological, biochemical (oxygen free radicals), etc., events in the development of gastroin‐ testinal mucosal injury and its prevention (Mózsik et al. 1992). Gastric mucosal damage can be produced by direct application of ethanol and indomethacin and it can be dose depend‐ ently prevented by the topical application of capsaicin in healthy volunteers (Mózsik et al., 2005a). Endogenous (increased production of gastric HCl in 4-hr pylorus-ligated rats) and exogenous (indomethacin given s.c. in a 4-hr experiment, without pylorus ligation) factors produced changes in functional status and damaged the gastric mucosa in rats. The immu‐ nohistochemical distribution of TRPV1, CGRP, and SP decreased significantly during this time; the levels of TRPV1 and CGRP returned to those of healthy controls dose dependently by application of omeprazole and omeprazole-like compounds, and no significant change was detected in the immunodistribution of SP (Mózsik et al. 1997b). There is no real explan‐ ation for the different expression rates of TRPV1 and mediators released from the sensory nerves. Similar differences in TRPV1, CGRP, and SP have been observed in many other con‐ ditions (in acute animal models and in patients with chronic gastrointestinal disorders). The following explanations have been suggested for these differences in expression of TRPV1 and mediators released from sensory nerves (Dömötör et al., 2007): (1) the regulation of SP differs from that of TRPV1 and CGRP regarding the development of chronic gastritis and (2) the sensitivity of SP regulation is lower than in the cases of TRPV1 and CGRP. The increased level of TRPV1 and CGRP is accepted as a signal of an increased defense mechanism pro‐ duced by capsaicin-sensitive afferent nerves in animal experiments (Mózsik et al. 2005a), and the topical application of capsaicin dose-dependently prevented the mucosal injury pro‐ duced by intragastrically applied ethanol and indomethacin in healthy human subjects (Mózsik et al. 2005b). These findings suggest that increased expression of TRPV1 and CGRP is associated with increased gastric mucosal protection. This gastric mucosal protective mechanism provided by capsaicin-sensitive afferent nerves exists in patients with chronic gastritis, and its extent does not depend on the presence or absence of *H. pylori* infection. In other words, the gastric mucosal protection produced by capsaicin-sensitive afferent nerves is one of the general defensive mechanisms against the different noxious agents that induce

Capsaicin-Sensitive Afferentation Represents a New Mucosal Defensive Neural Pathway System in the Gastric...

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69

The results of above mentioned animal and human observations indicated clearly the fol‐

1. Both acute and chronic gastritis can be induced by different reasons, like chemicals (drugs) or bacteria (most emphysized one H. Pylori). Both H. pylori positive and negative gastritis can be acute or chroinic. In case of patients with H.plyori negative gastritis, the therapeutic approach follows the traditional medical treatment, while H.pylori positive pa‐ tients with gastritis are suggested a rational eradtication treatment only (accepted by many

chronic gastritis.

**3. Conclusions**

lowing considerations:

international consensus meetings).

Explantaion of study group of the for the unchanged immunohistochemical distribution of TRPV1, CGRP and SP of gastric *H. pylori* positive chronic gastritis before vs. after eradica‐ tion treatment were as follows (Mozsik et al. 2011):

Kozlowski et al. (2011) conluded from their study on chronic gastritis: 1. Chronic supeficial gastritis coexists with significantly higher proliferative activity of gastric mucosal glandular epithelium, particulary in relation to the prepyloric area and 2. Changes of proliferative ac‐ tivity of gastric mucosal glandular epithelium are independent of age, histotopography and of *H. pylori* colonization.

Recent investigations showed that the expression of TRPV1 and CGRP increased significant‐ ly inthe epithelial layer of the gastric mucosa in patients with *H. pylori -* positive and -nega‐ tive chronic gastritis. The significant etiological role of *H. pylori* in the development of chronic gastritis has received great attention in the pertinent literature; for this reason, we compared the differences in TRPV1,CGRP, and SP expression in *H. pylori* positive or -nega‐ tive chronic gastritis, suggesting their etiological role in the development of chronic gastritis. No significant differences were obtained between *H. pylori-*positive and *H. pylori*-negative patients, but values of both groups with chronic gastritis differed significantly from those of histological intact mucosa of healthy human subjects. Results also indicated that chronic gastric inflammation is one of the general tissue reactions to different noxious agents, and *H. pylori* is only one of these in patients. It must be emphasized that clinicians are able to specif‐ ically demonstrate the presence of *H. pylori* (by UBT, rapid urease test, and specific histologi‐ cal staining), however, we have no specific methods for well demonstration of other (suggested) etiological factors.

In animal experiments, different noxious agents produce the same (or basically similar) pathological, biochemical (oxygen free radicals), etc., events in the development of gastroin‐ testinal mucosal injury and its prevention (Mózsik et al. 1992). Gastric mucosal damage can be produced by direct application of ethanol and indomethacin and it can be dose depend‐ ently prevented by the topical application of capsaicin in healthy volunteers (Mózsik et al., 2005a). Endogenous (increased production of gastric HCl in 4-hr pylorus-ligated rats) and exogenous (indomethacin given s.c. in a 4-hr experiment, without pylorus ligation) factors produced changes in functional status and damaged the gastric mucosa in rats. The immu‐ nohistochemical distribution of TRPV1, CGRP, and SP decreased significantly during this time; the levels of TRPV1 and CGRP returned to those of healthy controls dose dependently by application of omeprazole and omeprazole-like compounds, and no significant change was detected in the immunodistribution of SP (Mózsik et al. 1997b). There is no real explan‐ ation for the different expression rates of TRPV1 and mediators released from the sensory nerves. Similar differences in TRPV1, CGRP, and SP have been observed in many other con‐ ditions (in acute animal models and in patients with chronic gastrointestinal disorders). The following explanations have been suggested for these differences in expression of TRPV1 and mediators released from sensory nerves (Dömötör et al., 2007): (1) the regulation of SP differs from that of TRPV1 and CGRP regarding the development of chronic gastritis and (2) the sensitivity of SP regulation is lower than in the cases of TRPV1 and CGRP. The increased level of TRPV1 and CGRP is accepted as a signal of an increased defense mechanism pro‐ duced by capsaicin-sensitive afferent nerves in animal experiments (Mózsik et al. 2005a), and the topical application of capsaicin dose-dependently prevented the mucosal injury pro‐ duced by intragastrically applied ethanol and indomethacin in healthy human subjects (Mózsik et al. 2005b). These findings suggest that increased expression of TRPV1 and CGRP is associated with increased gastric mucosal protection. This gastric mucosal protective mechanism provided by capsaicin-sensitive afferent nerves exists in patients with chronic gastritis, and its extent does not depend on the presence or absence of *H. pylori* infection. In other words, the gastric mucosal protection produced by capsaicin-sensitive afferent nerves is one of the general defensive mechanisms against the different noxious agents that induce chronic gastritis.

#### **3. Conclusions**

mated chronic gastritis, and no significant change was obtained in SP levels. However, these immunohistochemical results did not differ in patients with *H. pylori* positive and negative chronic gastritis. Capsaicin-sensitive afferention did not differ before and after (6 weeks) successfull eradication treatment in patients with chronic *H. pylori* positive gastritis (mean‐ while the control biopsy was normal in 22% and in 78% indicated in moderated histological

Explantaion of study group of the for the unchanged immunohistochemical distribution of TRPV1, CGRP and SP of gastric *H. pylori* positive chronic gastritis before vs. after eradica‐

**1.** Six week time period (including the eradication treatment) is not enough time for the

**2.** The six-week time period (after eradication treatment) is probably not enought time for complete histologically recovery of chronic *H. pylori* positive infection in patients in

**3.** The *H. pylori* bacteria as etiological factors might represent only one of the factors caus‐

**4.** The immunohistological distribution (expression rate) of TRPV1, CGRP and SP are in‐ dependent ont the chronic gastritis produced by different physical, chemical, bactero‐ logical or immunological agents. However, the increased expression of TRPV1, CGRP is involved in the gastric mucosal damage and the normalization of these changes can be

obtained by other way as the classical eradication treatment (Lakner et al. 2011).

Kozlowski et al. (2011) conluded from their study on chronic gastritis: 1. Chronic supeficial gastritis coexists with significantly higher proliferative activity of gastric mucosal glandular epithelium, particulary in relation to the prepyloric area and 2. Changes of proliferative ac‐ tivity of gastric mucosal glandular epithelium are independent of age, histotopography and

Recent investigations showed that the expression of TRPV1 and CGRP increased significant‐ ly inthe epithelial layer of the gastric mucosa in patients with *H. pylori -* positive and -nega‐ tive chronic gastritis. The significant etiological role of *H. pylori* in the development of chronic gastritis has received great attention in the pertinent literature; for this reason, we compared the differences in TRPV1,CGRP, and SP expression in *H. pylori* positive or -nega‐ tive chronic gastritis, suggesting their etiological role in the development of chronic gastritis. No significant differences were obtained between *H. pylori-*positive and *H. pylori*-negative patients, but values of both groups with chronic gastritis differed significantly from those of histological intact mucosa of healthy human subjects. Results also indicated that chronic gastric inflammation is one of the general tissue reactions to different noxious agents, and *H. pylori* is only one of these in patients. It must be emphasized that clinicians are able to specif‐ ically demonstrate the presence of *H. pylori* (by UBT, rapid urease test, and specific histologi‐ cal staining), however, we have no specific methods for well demonstration of other

picture of gastritis) (Lakner et al. 2011).

68 Current Topics in Gastritis - 2012

tion treatment were as follows (Mozsik et al. 2011):

complete healing of chronic gastritis.

term of histology and immunohistology.

ing chronic gastritis (in term of histology);

of *H. pylori* colonization.

(suggested) etiological factors.

The results of above mentioned animal and human observations indicated clearly the fol‐ lowing considerations:

1. Both acute and chronic gastritis can be induced by different reasons, like chemicals (drugs) or bacteria (most emphysized one H. Pylori). Both H. pylori positive and negative gastritis can be acute or chroinic. In case of patients with H.plyori negative gastritis, the therapeutic approach follows the traditional medical treatment, while H.pylori positive pa‐ tients with gastritis are suggested a rational eradtication treatment only (accepted by many international consensus meetings).

Mózsik et al. and Lakner et al. (2011) have demonstrated that while chronic H. pylori pozi‐ tive gastritis heals entirely after a sucessful eradication therapy (which represents an impor‐ tant step to prevent development of gastric cancer), the extents of TRVP1, CGRP remained in increased level (in comparison to the normal gastric mucosa). This indicates that there is at least one other important defensive mechanism than antibiotic eradiction treatment, the capsaicin-sensivite afferention of vagal nerve (results clearly indicated that independent from H. pylori status and independent from eradication).

[2] Aijoka, H., Miyake, H., & Matsuura, N. (2000). Effect of FRG-8813, a new-type hista‐ mine H2-receptor antagonist, on the recurrence of gastric ulcer healing by drug treat‐

Capsaicin-Sensitive Afferentation Represents a New Mucosal Defensive Neural Pathway System in the Gastric...

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71

[3] Aijoka, H., Matsuura, N., & Miyake, H. (2002). High quality of ulcer healing in rats by lafutidine and new-tipe histamine H2-receptor antagonist: involvement of capsai‐

[4] Appelman HD(1994). Gastritis: terminology, etiology, and clinicopathological corre‐

[5] Buck SH, Burks TF.(1986). The Neuropharmacology of capsaicin: review of some re‐

[6] Caterina, Chumacher., Tominaga, M., Rosen, T. A., & Levine, Julius. D. (1997). The capsaicin receptor: a heat-activated ion channel in the pain pathway. *Nature*, 389,

[7] Dixon, M. F., Genta, R. M., Yardley, J. H., & Correa, P. (1996). Classification and grad‐ ing of gastritis: the updated Sydney system. Am J Surg Pathol , 20, 1161-1181.

[8] Dömötör, A., Peidl, Zs., Vincze, Á., Hunyady, B., Szolcsányi, J., Kereskay, L., Sze‐ keres, Gy., & Mózsik, Gy. (2005). Immunohistochemical distribution of vanilloid re‐ ceptor (TRPV1), calcitonin generelated peptide (CGRP) and substance-P (SP) in the gastrointestinal mucosa of patients with different gastrointestinal disorders. Inflam‐

[9] Dömötör, A., Kereskay, L., Szekeres, Gy., Hunyady, B., Szolcsányi, J., & Mózsik, Gy. (2006). Participation of capsaicin sensitive afferent nerves in the gastric mucosa of pa‐ tients with helicobacter pyloripositive or-negative chronic gastritis. Dig Dis Sci. , 52,

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[12] Holzer, P. Afferent nerve-mediated control of gastric mucosal blood flow and protec‐ tion. In: Costa M, Surrenti C, Gorini S, Maggi CA, Meli A, Eds. Sensory nerve and neuropeptides in gastroenterology. From basic science to clinical perspective. New

[13] Holzer, P. (1998). Neural emergency system in the stomach. *Gastroenterology*, 114,

[14] Holzer, P. (1999). Capsaicin cellular targets, mechanisms of action and selectivity for

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Generally it can be concluded that: 1. there can be different mucosal defensive mechanisms playing key role in treatment of H. plyori positive chronic gastritis: a. eradication treatment and b. stimulation of capsaicin sensitive afferentation; 2. eradication treatment is independ‐ ent from capsaicin-sensitive afferention (defensive) mechanism; 3. it should be considered to amend treatment protocols of H. pylori positive chronic gastritis (conventional eradication therapy and success control) with modification of functional state of capsaicin-sensitive af‐ ferentation. Medical effect of capsaicin for modification of capsacin-sensitive afferent nerves depends on its dosages, more clinical findngs are needed in the field, that observations are in progress (Mozsik et al. 2009b, 2011).

#### **Acknowledgements**

This chapter was supported by MEDICZIM Kft, Hungary and ALAPITVANY AZ EGESZ‐ SEG UGYEBEN DOLGOZOKERT, Hungary.

#### **Author details**

Jozsef Czimmer1\*, Imre Laszló Szabo1 , Janos Szolcsanyi2 and Gyula Mozsik1

\*Address all correspondence to<EMAIL_ADDRESS>

#### **References**

[1] Abdel-Salam, O. M. E., Debreceni, A., & Mózsik, Gy. (1999). Capsaicinsensitive affer‐ ent sensory nerves in modulating gastric mucosal defense against noxious agents. J Physiol Paris , 93, 443-454.

[2] Aijoka, H., Miyake, H., & Matsuura, N. (2000). Effect of FRG-8813, a new-type hista‐ mine H2-receptor antagonist, on the recurrence of gastric ulcer healing by drug treat‐ ment. Pharmacology. , 61, 83-90.

Mózsik et al. and Lakner et al. (2011) have demonstrated that while chronic H. pylori pozi‐ tive gastritis heals entirely after a sucessful eradication therapy (which represents an impor‐ tant step to prevent development of gastric cancer), the extents of TRVP1, CGRP remained in increased level (in comparison to the normal gastric mucosa). This indicates that there is at least one other important defensive mechanism than antibiotic eradiction treatment, the capsaicin-sensivite afferention of vagal nerve (results clearly indicated that independent

Generally it can be concluded that: 1. there can be different mucosal defensive mechanisms playing key role in treatment of H. plyori positive chronic gastritis: a. eradication treatment and b. stimulation of capsaicin sensitive afferentation; 2. eradication treatment is independ‐ ent from capsaicin-sensitive afferention (defensive) mechanism; 3. it should be considered to amend treatment protocols of H. pylori positive chronic gastritis (conventional eradication therapy and success control) with modification of functional state of capsaicin-sensitive af‐ ferentation. Medical effect of capsaicin for modification of capsacin-sensitive afferent nerves depends on its dosages, more clinical findngs are needed in the field, that observations are

This chapter was supported by MEDICZIM Kft, Hungary and ALAPITVANY AZ EGESZ‐

, Janos Szolcsanyi2

[1] Abdel-Salam, O. M. E., Debreceni, A., & Mózsik, Gy. (1999). Capsaicinsensitive affer‐ ent sensory nerves in modulating gastric mucosal defense against noxious agents. J

2 Department of Pharmacology and Pharmacotherapy, University of Pécs, Hungary

and Gyula Mozsik1

from H. pylori status and independent from eradication).

in progress (Mozsik et al. 2009b, 2011).

SEG UGYEBEN DOLGOZOKERT, Hungary.

\*Address all correspondence to<EMAIL_ADDRESS>
1 First Department of Medicine, University of Pécs, Hungary

Jozsef Czimmer1\*, Imre Laszló Szabo1

Physiol Paris , 93, 443-454.

**Acknowledgements**

70 Current Topics in Gastritis - 2012

**Author details**

**References**

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[27] Mózsik, Gy., Nagy, L., Pár, A., & Rainsford, . (1997b). Eds. Cell Injury and Protection in the Gastrointestinal Tract: from Basic Science to Clinical Perspectives. Dordrecht:

Capsaicin-Sensitive Afferentation Represents a New Mucosal Defensive Neural Pathway System in the Gastric...

http://dx.doi.org/10.5772/52883

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74 Current Topics in Gastritis - 2012

**Section 5**

**Diagnostic Backgrounds**

**Diagnostic Backgrounds**

**Chapter 5**

**The Genetic and Epigenetic Bases of Gastritis**

Gastritis, the inflammation of the mucosal layer of the stomach, is a major clinical entity due to its association with gastric cancer and peptic ulcer disease. The primary cause of gastritis is the infection with the microaerofilic gram negative Helicobacter pylori that during the early phases elicits an acute inflammatory response which eventually evolves to a longstanding chronic gastritis (Ruggiero 2012). In the case of gastric cancer development, chronic gastritis is the first step of the so-called multistep cascade of gastric cancer. This sequence includes the non-atrophic chronic gastritis, multifocal atrophic gastritis, intestinal metapla‐ sia, dysplasia and invasive carcinoma as were described in detail by Correa as the "human model of gastric carcinogenesis" (Fig. 1) (Correa et al. 2007). This multistep model hypothe‐ sizes that the sequence of these lesions reflects a dynamic process from a naive inflammation caused by H. pylori infection to a fully malignant neoplasm of the stomach (Correa et al. 1976; Cuello et al. 1976; Haenszel et al. 1976; Correa et al. 1990). Independent epidemiologi‐ cal studies have confirmed that non-atrophic, atrophic, intestinal metaplasia and dysplasia are all linked through a sequential cause-effect relationship, thus supporting the concept of a human model for gastric carcinogenesis (Ohata et al. 2004). However, the risk of malignant transformation of these lesions is poorly defined. Long-term follow-up studies have shown a risk from 10% to 17% in the case of dysplasia (Saraga et al. 1987; Coma del Corral et al. 1990; Koch et al. 1990; Whiting et al. 2002; Rugge et al. 2003). For intestinal metaplasia, the risk assessment has conflicting results and therefore a limited clinical value (Ramesar et al. 1987; Silva et al. 1990; Rokkas et al. 1991; Conchillo et al. 2001; Vannella et al. 2012). The re‐ cently developed Operative Link for Gastritis Assessment (OLGA) staging system (Rugge et al. 2007), through the evaluation of the extension and site of the atrophic changes, is an at‐ tempt to evaluate the risk of chronic gastritis to progress to intestinal metaplasia and gastric cancer (Rugge et al. 2008; Capelle et al. 2010; Rugge et al. 2010). In this scenario, the identifi‐

> © 2013 Corvalan et al.; licensee InTech. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

and reproduction in any medium, provided the original work is properly cited.

© 2013 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution,

Alejandro H. Corvalan, Gonzalo Carrasco and

Additional information is available at the end of the chapter

Kathleen Saavedra

**1. Introduction**

http://dx.doi.org/10.5772/53131

### **The Genetic and Epigenetic Bases of Gastritis**

Alejandro H. Corvalan, Gonzalo Carrasco and Kathleen Saavedra

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/53131

#### **1. Introduction**

Gastritis, the inflammation of the mucosal layer of the stomach, is a major clinical entity due to its association with gastric cancer and peptic ulcer disease. The primary cause of gastritis is the infection with the microaerofilic gram negative Helicobacter pylori that during the early phases elicits an acute inflammatory response which eventually evolves to a longstanding chronic gastritis (Ruggiero 2012). In the case of gastric cancer development, chronic gastritis is the first step of the so-called multistep cascade of gastric cancer. This sequence includes the non-atrophic chronic gastritis, multifocal atrophic gastritis, intestinal metapla‐ sia, dysplasia and invasive carcinoma as were described in detail by Correa as the "human model of gastric carcinogenesis" (Fig. 1) (Correa et al. 2007). This multistep model hypothe‐ sizes that the sequence of these lesions reflects a dynamic process from a naive inflammation caused by H. pylori infection to a fully malignant neoplasm of the stomach (Correa et al. 1976; Cuello et al. 1976; Haenszel et al. 1976; Correa et al. 1990). Independent epidemiologi‐ cal studies have confirmed that non-atrophic, atrophic, intestinal metaplasia and dysplasia are all linked through a sequential cause-effect relationship, thus supporting the concept of a human model for gastric carcinogenesis (Ohata et al. 2004). However, the risk of malignant transformation of these lesions is poorly defined. Long-term follow-up studies have shown a risk from 10% to 17% in the case of dysplasia (Saraga et al. 1987; Coma del Corral et al. 1990; Koch et al. 1990; Whiting et al. 2002; Rugge et al. 2003). For intestinal metaplasia, the risk assessment has conflicting results and therefore a limited clinical value (Ramesar et al. 1987; Silva et al. 1990; Rokkas et al. 1991; Conchillo et al. 2001; Vannella et al. 2012). The re‐ cently developed Operative Link for Gastritis Assessment (OLGA) staging system (Rugge et al. 2007), through the evaluation of the extension and site of the atrophic changes, is an at‐ tempt to evaluate the risk of chronic gastritis to progress to intestinal metaplasia and gastric cancer (Rugge et al. 2008; Capelle et al. 2010; Rugge et al. 2010). In this scenario, the identifi‐

© 2013 Corvalan et al.; licensee InTech. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. © 2013 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

cation of molecular bases in the multistep process of gastric carcinogenesis is highly relevant since it will contribute greatly to the risk assessments of the precursor lesions of gastric can‐ cer. In this chapter we will primarily attempt to summarize and integrate our current knowledge of the genetic as well as epigenetic bases of the dynamic process of chronic gas‐ tritis, as well as the other entities of the so-called multistep cascade of gastric cancer.

genotype enhance the production of IL-1B, which is associated with an increased risk of hy‐ pochlorhydria induced by H. pylori. Because IL-1B is an important pro-inflammatory cyto‐ kine and a powerful inhibitor of gastric acid secretion, it might explain the higher risk of development of gastric cancer in chronic gastritis - H. pylori infected patients (El-Omar et al. 2000; Hwang et al. 2002; Yuzhalin 2011). The presence of polymorphisms in the non-coding but regulatory regions may also alter the levels of gene transcription and therefore the sus‐ ceptibility to gastric cancer. In this sense, single A/T SNP at position -251 from the transcrip‐ tion start site in the promoter region of the IL-8 gene is one of the best examples. The presence of the -251A allele tended to be associated with an increase in IL-8 production and has been reported that this allele is associated with an increased risk for gastric cancer at car‐ dia location (Hull et al. 2000; Xue et al. 2012). It could be extrapolated that IL-8 -251A allele may increase the risk of developing cancer through the elevation of its IL-8 expression. Me‐ diators of the innate immune response, which provide first line of host defense against harmful pathogens are also involved in the dynamics of chronic gastritis. A good example for this type of inflammatory response are the Toll-like receptors (TLRs) (El-Omar et al. 2008). It has been reported that both, the 22-bp nucleotide deletion (-196 to –174 del) in the promoter region of the TLR-2 gene and the +896A/G and +1196C/T polymorphisms (Asp299Gly and Thr399Ile) in the coding region of the TLR-4 gene, promotes a rapidly pro‐ gressive chronic gastritis (de Oliveira et al. 2012). Since it is well known that not all H. pylori are equally associated with the risk of development of gastric cancer, the combination of bacterial and inflammatory host genotypes have been explored. Figueiredo et al (2002) have shown that the infection with aggressive cagA-positive strains of H. pylori were associated with an increased risk of gastric cancer. In addition, the homozygous IL-1B-511\*T carriers (IL-1B-511\*T/\*T or IL-1B-511\*T/\*C) and the short allele of IL-1RN (IL-1RN\*2/\*2) also had an increased susceptibility. However, for each combination of bacterial/host genotype, the odds of having gastric cancer were greatest in those with both bacterial and host high-risk geno‐

The Genetic and Epigenetic Bases of Gastritis

http://dx.doi.org/10.5772/53131

81

types.

ferentiated tumors (Table 1).

Polymorphisms in genes associated with detoxification enzymes

Members of the cytochrome P-450 superfamily belong to the detoxification enzymes. One of the members of this family is the CYP2E1, a naturally ethanol-inducible enzyme involved in the metabolic activation of low molecular weight compounds such as N-nitrosamines (Boc‐ cia et al. 2007). Functional CYP2E1 polymorphisms in the 50-flanking region (PstI, RsaI) alter the transcriptional activity of the gene. A meta-analysis by Boccia et al (2007) suggests that the CYP2E1 PstI/RsaI polymorphism may be a risk factor for gastric cancer in asiatic popula‐ tions and a synergic relation with tobacco-related detoxification Glutathione S-transferase (GST) genes (see below) may account for a proportion of these cases. GSTs and the polymor‐ phic arylamine N-acetyltransferases (NAT1 and NAT2) are another type of detoxification enzymes that metabolize tobacco-related carcinogens. Zendehdel et al (2009) reported a weak linkage between the GSTP1 Ile105Val polymorphism and the risk for gastric cancer at cardia location (OR = 1.4; 95% CI 0.9-2.1). On the other hand, Katoh et al (2000) found that the NAT1\*10 allele should be considered a risk factor among heavy smokers with well-dif‐

**Figure 1.** Multistep cascade of gastric cancer. This sequence begins with the infection of H.pylori to sequential steps of the precancerous cascades. Taken from Correa & Piazuelo J Dig Dis. 2012;13:2–9.

#### **2. The genetic bases of gastritis**

Human allelic variations at single nucleotide polymorphisms (SNP) are involved at different stages of gastric carcinogenesis. Accordingly, the dynamics of chronic gastritis might be as‐ sociated with specific allelic variants. These variants, recognized as polymorphisms when occur with a frequency of >1% in the normal population, affects mostly genes of the inflam‐ matory response genes, detoxification enzymes, and cancer-related procesess (Gonzalez et al. 2002).

Polymorphisms in inflammatory response genes

Two clear examples of polymorphisms associated with inflammatory response are interleu‐ kin-1 gene cluster and Toll-like receptors (TLRs). As shown in Table 1, Interleukin-1-beta (IL-1B) -31 T genotype and interleukin-1-receptor antagonist (IL1RN) IVS 86 bp VNTR, 2/2

genotype enhance the production of IL-1B, which is associated with an increased risk of hy‐ pochlorhydria induced by H. pylori. Because IL-1B is an important pro-inflammatory cyto‐ kine and a powerful inhibitor of gastric acid secretion, it might explain the higher risk of development of gastric cancer in chronic gastritis - H. pylori infected patients (El-Omar et al. 2000; Hwang et al. 2002; Yuzhalin 2011). The presence of polymorphisms in the non-coding but regulatory regions may also alter the levels of gene transcription and therefore the sus‐ ceptibility to gastric cancer. In this sense, single A/T SNP at position -251 from the transcrip‐ tion start site in the promoter region of the IL-8 gene is one of the best examples. The presence of the -251A allele tended to be associated with an increase in IL-8 production and has been reported that this allele is associated with an increased risk for gastric cancer at car‐ dia location (Hull et al. 2000; Xue et al. 2012). It could be extrapolated that IL-8 -251A allele may increase the risk of developing cancer through the elevation of its IL-8 expression. Me‐ diators of the innate immune response, which provide first line of host defense against harmful pathogens are also involved in the dynamics of chronic gastritis. A good example for this type of inflammatory response are the Toll-like receptors (TLRs) (El-Omar et al. 2008). It has been reported that both, the 22-bp nucleotide deletion (-196 to –174 del) in the promoter region of the TLR-2 gene and the +896A/G and +1196C/T polymorphisms (Asp299Gly and Thr399Ile) in the coding region of the TLR-4 gene, promotes a rapidly pro‐ gressive chronic gastritis (de Oliveira et al. 2012). Since it is well known that not all H. pylori are equally associated with the risk of development of gastric cancer, the combination of bacterial and inflammatory host genotypes have been explored. Figueiredo et al (2002) have shown that the infection with aggressive cagA-positive strains of H. pylori were associated with an increased risk of gastric cancer. In addition, the homozygous IL-1B-511\*T carriers (IL-1B-511\*T/\*T or IL-1B-511\*T/\*C) and the short allele of IL-1RN (IL-1RN\*2/\*2) also had an increased susceptibility. However, for each combination of bacterial/host genotype, the odds of having gastric cancer were greatest in those with both bacterial and host high-risk geno‐ types.

#### Polymorphisms in genes associated with detoxification enzymes

cation of molecular bases in the multistep process of gastric carcinogenesis is highly relevant since it will contribute greatly to the risk assessments of the precursor lesions of gastric can‐ cer. In this chapter we will primarily attempt to summarize and integrate our current knowledge of the genetic as well as epigenetic bases of the dynamic process of chronic gas‐

**Figure 1.** Multistep cascade of gastric cancer. This sequence begins with the infection of H.pylori to sequential steps of

Human allelic variations at single nucleotide polymorphisms (SNP) are involved at different stages of gastric carcinogenesis. Accordingly, the dynamics of chronic gastritis might be as‐ sociated with specific allelic variants. These variants, recognized as polymorphisms when occur with a frequency of >1% in the normal population, affects mostly genes of the inflam‐ matory response genes, detoxification enzymes, and cancer-related procesess (Gonzalez et

Two clear examples of polymorphisms associated with inflammatory response are interleu‐ kin-1 gene cluster and Toll-like receptors (TLRs). As shown in Table 1, Interleukin-1-beta (IL-1B) -31 T genotype and interleukin-1-receptor antagonist (IL1RN) IVS 86 bp VNTR, 2/2

the precancerous cascades. Taken from Correa & Piazuelo J Dig Dis. 2012;13:2–9.

**2. The genetic bases of gastritis**

80 Current Topics in Gastritis - 2012

Polymorphisms in inflammatory response genes

al. 2002).

tritis, as well as the other entities of the so-called multistep cascade of gastric cancer.

Members of the cytochrome P-450 superfamily belong to the detoxification enzymes. One of the members of this family is the CYP2E1, a naturally ethanol-inducible enzyme involved in the metabolic activation of low molecular weight compounds such as N-nitrosamines (Boc‐ cia et al. 2007). Functional CYP2E1 polymorphisms in the 50-flanking region (PstI, RsaI) alter the transcriptional activity of the gene. A meta-analysis by Boccia et al (2007) suggests that the CYP2E1 PstI/RsaI polymorphism may be a risk factor for gastric cancer in asiatic popula‐ tions and a synergic relation with tobacco-related detoxification Glutathione S-transferase (GST) genes (see below) may account for a proportion of these cases. GSTs and the polymor‐ phic arylamine N-acetyltransferases (NAT1 and NAT2) are another type of detoxification enzymes that metabolize tobacco-related carcinogens. Zendehdel et al (2009) reported a weak linkage between the GSTP1 Ile105Val polymorphism and the risk for gastric cancer at cardia location (OR = 1.4; 95% CI 0.9-2.1). On the other hand, Katoh et al (2000) found that the NAT1\*10 allele should be considered a risk factor among heavy smokers with well-dif‐ ferentiated tumors (Table 1).

#### Polymorphisms of cancer-related genes

In cancer-related processes, polymorphisms have been reported in the promoter region of the MET gene, a crucial gene to multiple oncogenic pathways and metastatic behavior (Gherardi et al. 2012). Sequencing of the promoter region of MET revealed some alterations scattered in a proportion of clinical samples of gastric cancer. The most common substitu‐ tions were -304C>A and 206C>G. The presence of these SNPs altered the junction sites for putative transcription factors such as Sp1 and AP-1/AP-2. As a consequence, the transcrip‐ tion of MET was constantly being activated (Trzyna et al. 2012). Another example is the polymorphism -160 A at the promoter region of the E-cadherin gene (CDH1), which has been associated with the DNA hypermethylation (see below) of the promoter region (Borges Bdo et al. 2010). In a Japanase study, 117 cases of gastric cancer with H. pylori-induced chronic gastritis were compared with 116 cancer-free yet H. pylori-induced chronic gastritis controls. It was found that the Pro/Pro allele of CDH-1 was associated with an increased risk of developing diffuse-type of gastric cancer compared to the Arg/Arg (Hiyama et al. 2002; Zhou et al. 2007). Finally, other polymorphims of cancer-related genes were reported to show significant associations with gastric cancer risk including EGFR, VEGF and p53 (Ta‐ hara et al. 2009). Taken together, data presented here suggest that allelic variations at regula‐ tory or coding regions will affect gene expression patterns and modify the balance towards a more rapid progression of chronic gastritis and other preneoplastic lesions of the multistep cascade of gastric cancer (Fig. 2).

**Gene Variation Allelic SNP rs number Effect Reference**

IL-8 SNP -251 A/T rs4073 Increased IL-8 production. (Xue et al.,

TLR4 SNP + 896 A/G rs4986790 al, 2012)

homozygote not applicable Risk for gastric cancer among

SNP 206 C>G undefined 2012)

**Table 1.** Selected single nucleotide polymorphisms (SNP) in inflammatory response genes, detoxification enzymes, and cancer-related processes associated to increase susceptibility of gastric cancer. The sign (-) indicates that the SNP is located in the direction of the promoter region, the sign (+) indicates that the SNP is located in the direction of the coding region, the number indicates the nucleotide positionfrom TSS and the first nucleotide is substituted (/) by the

Epigenetic processes control the packaging and function of the human genome, and contrib‐ ute to normal development and disease (Callinan et al. 2006). Epigenetic mechanisms such as DNA methylation, histone modifications and microRNAs are important events toward regulating gene expression for the biology and disease of the gastrointestinal tract. In partic‐ ular, DNA methylation, a process in which cytosines acquire a methyl group in the 5' posi‐ tion only if they are followed by a guanine (Corvalán et al. 2010), virtually affects all of the pathways in the cellular network, such as DNA repair, cell cycle, and apoptosis (Esteller et

and inhibit gastric acid secretion.

The Genetic and Epigenetic Bases of Gastritis

http://dx.doi.org/10.5772/53131

Reported associated with the pathologically intestinal-type gastric cancer or anatomically cardia-type gastric cancer.

the Southeastern Brazilian

Increased risk of esophageal squamous cell carcinoma and tended to be weakly, positively linked to cardia cancer

Risk factor among the welldifferentiated type of tumors of

Associated with DNA methylation linking genetic and epigenetics fields in the pathogenesis of

Activate the transcription. (Trzyna et al.,

population.

gastric cancer

gastritis.

Asians

(Yuzhalin et al., 2011)

83

2012)

(Ni et al., 2012)

(de Oliveira et

(Boccia et al., 2006)

(Zendehdel et al., 2009)

(Katoh et al., 2000)

(Borges Bdo et al., 2010)

IL1B SNP -31 T/C rs1143627 Increased IL-1beta production

TLR2 Deletion -196 to -174del not applicable Susceptibility to gastric cancer in

IL-10 SNP -1082 A/G rs1800896

GSTP1 SNP +562 A/G rs1695

NAT RFLP NAT1\*10 allele not applicable

MET SNP -304C> A undefined

CDH1 SNP -160 C/A rs16260

**3. Epigenetic bases of gastritis**

CYP2E1 c2 allele

CYP2E1 RFLP

second.

**Figure 2.** Allelic variations at regulatory regions will affect gene expression patterns. (A) Single nucleotide polymor‐ phisms (SNP) in promoter region can alter the binding of transcription factors required for the expression of a gene. (B) These variations may increase or decrease the expression of the affected gene, which eventually can increase the risk of developing cancer.

**Table 1.** Selected single nucleotide polymorphisms (SNP) in inflammatory response genes, detoxification enzymes, and cancer-related processes associated to increase susceptibility of gastric cancer. The sign (-) indicates that the SNP is located in the direction of the promoter region, the sign (+) indicates that the SNP is located in the direction of the coding region, the number indicates the nucleotide positionfrom TSS and the first nucleotide is substituted (/) by the second.

#### **3. Epigenetic bases of gastritis**

Polymorphisms of cancer-related genes

82 Current Topics in Gastritis - 2012

cascade of gastric cancer (Fig. 2).

risk of developing cancer.

In cancer-related processes, polymorphisms have been reported in the promoter region of the MET gene, a crucial gene to multiple oncogenic pathways and metastatic behavior (Gherardi et al. 2012). Sequencing of the promoter region of MET revealed some alterations scattered in a proportion of clinical samples of gastric cancer. The most common substitu‐ tions were -304C>A and 206C>G. The presence of these SNPs altered the junction sites for putative transcription factors such as Sp1 and AP-1/AP-2. As a consequence, the transcrip‐ tion of MET was constantly being activated (Trzyna et al. 2012). Another example is the polymorphism -160 A at the promoter region of the E-cadherin gene (CDH1), which has been associated with the DNA hypermethylation (see below) of the promoter region (Borges Bdo et al. 2010). In a Japanase study, 117 cases of gastric cancer with H. pylori-induced chronic gastritis were compared with 116 cancer-free yet H. pylori-induced chronic gastritis controls. It was found that the Pro/Pro allele of CDH-1 was associated with an increased risk of developing diffuse-type of gastric cancer compared to the Arg/Arg (Hiyama et al. 2002; Zhou et al. 2007). Finally, other polymorphims of cancer-related genes were reported to show significant associations with gastric cancer risk including EGFR, VEGF and p53 (Ta‐ hara et al. 2009). Taken together, data presented here suggest that allelic variations at regula‐ tory or coding regions will affect gene expression patterns and modify the balance towards a more rapid progression of chronic gastritis and other preneoplastic lesions of the multistep

**Figure 2.** Allelic variations at regulatory regions will affect gene expression patterns. (A) Single nucleotide polymor‐ phisms (SNP) in promoter region can alter the binding of transcription factors required for the expression of a gene. (B) These variations may increase or decrease the expression of the affected gene, which eventually can increase the Epigenetic processes control the packaging and function of the human genome, and contrib‐ ute to normal development and disease (Callinan et al. 2006). Epigenetic mechanisms such as DNA methylation, histone modifications and microRNAs are important events toward regulating gene expression for the biology and disease of the gastrointestinal tract. In partic‐ ular, DNA methylation, a process in which cytosines acquire a methyl group in the 5' posi‐ tion only if they are followed by a guanine (Corvalán et al. 2010), virtually affects all of the pathways in the cellular network, such as DNA repair, cell cycle, and apoptosis (Esteller et al. 2002). Furthermore, a growing body of evidence has shown that aberrant DNA methyla‐ tion (DNA hypermethylation or DNA hypomethlation) is an early event in carcinogenesis (Jones et al. 2002; Herman et al. 2003; Kopelovich et al. 2003). In addition, aberrant DNA methylation has been recently considered to be an excellent candidate to explain how cer‐ tain environmental factors may increase the risk of developing cancer (Chan et al. 2006). Ac‐ cordingly, an emerging catalog of cancer-related genes inactivated by DNA hypermethylation in gastric cancer has been established (Hamilton et al. 2006; Wu et al. 2006; Bernal et al. 2008). However, there are limited reports on DNA hypermethylation anal‐ ysis in precursor lesions of gastric cancer. Kang et al. (2001) tested five genes (p16, hMLH1, DAP-kinase, THBS1, and TIMP-3) in precancerous conditions to identify three different classes of hypermethylated genes. Hypermethylation of DAP-kinase was found in all pre‐ malignant conditions, whereas hMLH1 and p16 were preferentially methylated in intestinal metaplasia (6.3% and 2.1%, respectively), adenomas (9.8% and 11.5%, respectively) and gas‐ tric cancer (20.3% and 42.2%, respectively). THBS-1 and TIMP-3 were also hypermethylated at a similar frequency in all premalignant conditions, but showed a marked increase from chronic gastritis to intestinal metaplasia (10.1% & 34.7% and 14.5% & 36.7%, respectively; P < 0.05), as well as from adenomas to carcinomas (28.3% & 48.4% and 26.7% & 57.4% respec‐ tively). Another study including 11 genes (COX-2, DAP-kinase, E-cadherin, GSTP1, MGMT, hMLH1, p14, p16, THBS1, TIMP3, and RASSF1A) and 268 premalignant conditions (Kang et al. 2003) identified specific patterns of aberrant DNA methylation associated to aging. Spe‐ cifically five genes (DAP-kinase, E-cadherin, p14, THBS1, and TIMP-3) showed a progres‐ sive increase in aberrant methylation frequency as a function of aging, whereas the other genes (COX-2, GSTP1, MGMT, hMLH1, p16, and RASSF1A) were rarely methylated. Male patients showed higher numbers of hypermethylated genes than females (3.2 vs. 2.1, respec‐ tively, P = 0.002) and cases with severe intestinal metaplasia also showed a higher frequency of aberrantly methylated genes. Taken together, these findings suggest that hypermethyla‐ tion occurs early in the multistep gastric carcinogenesis and that accumulate during this process. In addition, age and gender are closely associated with increased frequency of aber‐ rant DNA methylation. A further study demonstrated that DNA methylation of E-cadherin was associated with H. pylori infection (p=0.002) in an independent way of age and/or gas‐ tritis (Chan et al. 2003). Similarly, Maekita et al. (2006) carried out a more detailed analysis of the effect of H. pylori infection in the progression of aberrant DNA methylation in chronic gastritis. These authors collected gastric mucosa samples from 154 healthy volunteers (56 H. pylori negative and 98 H. pylori positive) and from 72 cases with gastric cancers (29 H. pylo‐ ri negative and 43 H. pylori positive). Among the healthy volunteers, methylation levels were 5.4- to 303-fold higher in H. pylori positive subjects than in H. pylori negative ones (P < 0.0001). Particularly, methylation levels of the LOX, HAND1 and THBD at their promoter regions were identified in H. pylori-positive individuals. Among H. pylori-negative individ‐ uals, methylation levels were 2.2- to 32-fold higher in gastric cancer cases than in age-match‐ ed healthy volunteers. These findings suggest that H. pylori infection potently induces aberrant DNA methylation of several genes beyond E-cadherin, and that methylation levels of specific genes seemed to reflect the risk of gastric cancer in H. pylori-negative subjects. A recent quantitative analysis of the promoter region of a novel gene Reprimo (RPRM), per‐

formed in Colombian residents from areas with high and low incidence of gastric cancer, demonstrated an association with virulent factors such as cagA and vacA s1/m1 regions of H. pylori strains. This data suggests that certain strains of H. pylori are associated with aber‐ rant DNA methylation on specific genes (Schneider et al. 2010). Interestingly, we have shown that Reprimo is not only found in gastric mucosa but also in the plasma of gastric cancer patients (Bernal et al. 2008). Thus, hypermethylated circulating DNA offers the op‐ portunity for non-invasive detection of gastric cancer and premalignant gastritis (Corvalán et al. 2010; Sapari et al. 2012). As mentioned before, an aberrant of DNA methylation could be considered as a candidate to explain how environmental factors increase the susceptibili‐ ty of gastric cancer. One approach to explain this phenomena could be through the evalua‐ tion of the effect of the eradication of H. pylori in the pattern of DNA methylation of the gastric mucosa. Chen et al (2003) evaluate the presence of DNA methylation of E-cadherin promoter regions before the erradication of H. pylori and after 6 weeks of treatment. DNA methylation of E-cadherin was detected in 46% (19/41) and 17% (7/41) of both untreated and treated patients, respectively. Mucosal biopsy showed chronic inactive gastritis in 35 pa‐ tients, intestinal metaplasia in one patient and normal mucosa in five patients after the treat‐ ment. This data suggests that H. pylori eradication therapy could reverse aberrant DNA methylation in patients with chronic gastritis. Similar results were reported by Leung et al. (2006), although they evaluated mucosal biopsies from the antrum and corpus of H. pyloriinfected subjects at the baseline, and after one year of successful H. pylori eradication. In ad‐ dition, these authors identified a significant reduction in the methylated density of the promoter region of the E-cadherin gene (Leung et al. 2006). Taken together, both reports are clear examples of how the environment affects DNA methylation and shows that the eradi‐ cation of H. pylori infection reverses E-cadherin promoter hypermethylation. Although less explored, DNA hypomethylation can also be involved in the risk of developing cancer. The concept of DNA hypomethylation, although identified 30 years ago (Christman et al. 1977), was overlooked in preference to DNA hypermethylation. Recently, gene activation by pro‐ moter hypomethylation has been rediscovered (Ichinose et al. 1988; Cravo et al. 1996; Fang et al. 1996; Akiyama et al. 2003; Cho et al. 2003; Mesquita et al. 2003; Oshimo et al. 2003; Chalitchagorn et al. 2004; Honda et al. 2004; Kaneda et al. 2004; Yanagawa et al. 2004; Jung et al. 2005; Nishigaki et al. 2005). Emerging data suggests that global DNA hypomethylation is thought to occur during the early stages of gastric carcinogenesis (Goelz et al. 1985; Cravo et al. 1996; Narayan et al. 1998; Lin et al. 2001; Bariol et al. 2003) but hypomethylation of indi‐ vidual genes, such as MAGE, synuclein-alpha, MUC2, maspin, CAGE and family A melano‐ ma antigen (Akiyama et al. 2003; Cho et al. 2003; Mesquita et al. 2003; Honda et al. 2004; Yanagawa et al. 2004; Jung et al. 2005) is thought to occur during the advanced stages. Inter‐ estingly, overexpression of well known oncogenes, such as MYC, HRAS1 and cyclin D2 has been linked to hypomethylation in their promoter region, suggesting an inverse correlation between methylation and gene expression (Fang et al. 1996; Oshimo et al. 2003). Taken to‐ gether, this data suggests that promoter hypomethylation is an underlying mechanism of gene activation that should be explored in gastric carcinogenesis. In these sense, recent work from Daskalos et al (2011) has shown that the overexpression of p73, a well stablish cancerrelated gene associated with apoptosis and DNA repair (Zaika et al. 2011), might be associ‐

The Genetic and Epigenetic Bases of Gastritis

http://dx.doi.org/10.5772/53131

85

formed in Colombian residents from areas with high and low incidence of gastric cancer, demonstrated an association with virulent factors such as cagA and vacA s1/m1 regions of H. pylori strains. This data suggests that certain strains of H. pylori are associated with aber‐ rant DNA methylation on specific genes (Schneider et al. 2010). Interestingly, we have shown that Reprimo is not only found in gastric mucosa but also in the plasma of gastric cancer patients (Bernal et al. 2008). Thus, hypermethylated circulating DNA offers the op‐ portunity for non-invasive detection of gastric cancer and premalignant gastritis (Corvalán et al. 2010; Sapari et al. 2012). As mentioned before, an aberrant of DNA methylation could be considered as a candidate to explain how environmental factors increase the susceptibili‐ ty of gastric cancer. One approach to explain this phenomena could be through the evalua‐ tion of the effect of the eradication of H. pylori in the pattern of DNA methylation of the gastric mucosa. Chen et al (2003) evaluate the presence of DNA methylation of E-cadherin promoter regions before the erradication of H. pylori and after 6 weeks of treatment. DNA methylation of E-cadherin was detected in 46% (19/41) and 17% (7/41) of both untreated and treated patients, respectively. Mucosal biopsy showed chronic inactive gastritis in 35 pa‐ tients, intestinal metaplasia in one patient and normal mucosa in five patients after the treat‐ ment. This data suggests that H. pylori eradication therapy could reverse aberrant DNA methylation in patients with chronic gastritis. Similar results were reported by Leung et al. (2006), although they evaluated mucosal biopsies from the antrum and corpus of H. pyloriinfected subjects at the baseline, and after one year of successful H. pylori eradication. In ad‐ dition, these authors identified a significant reduction in the methylated density of the promoter region of the E-cadherin gene (Leung et al. 2006). Taken together, both reports are clear examples of how the environment affects DNA methylation and shows that the eradi‐ cation of H. pylori infection reverses E-cadherin promoter hypermethylation. Although less explored, DNA hypomethylation can also be involved in the risk of developing cancer. The concept of DNA hypomethylation, although identified 30 years ago (Christman et al. 1977), was overlooked in preference to DNA hypermethylation. Recently, gene activation by pro‐ moter hypomethylation has been rediscovered (Ichinose et al. 1988; Cravo et al. 1996; Fang et al. 1996; Akiyama et al. 2003; Cho et al. 2003; Mesquita et al. 2003; Oshimo et al. 2003; Chalitchagorn et al. 2004; Honda et al. 2004; Kaneda et al. 2004; Yanagawa et al. 2004; Jung et al. 2005; Nishigaki et al. 2005). Emerging data suggests that global DNA hypomethylation is thought to occur during the early stages of gastric carcinogenesis (Goelz et al. 1985; Cravo et al. 1996; Narayan et al. 1998; Lin et al. 2001; Bariol et al. 2003) but hypomethylation of indi‐ vidual genes, such as MAGE, synuclein-alpha, MUC2, maspin, CAGE and family A melano‐ ma antigen (Akiyama et al. 2003; Cho et al. 2003; Mesquita et al. 2003; Honda et al. 2004; Yanagawa et al. 2004; Jung et al. 2005) is thought to occur during the advanced stages. Inter‐ estingly, overexpression of well known oncogenes, such as MYC, HRAS1 and cyclin D2 has been linked to hypomethylation in their promoter region, suggesting an inverse correlation between methylation and gene expression (Fang et al. 1996; Oshimo et al. 2003). Taken to‐ gether, this data suggests that promoter hypomethylation is an underlying mechanism of gene activation that should be explored in gastric carcinogenesis. In these sense, recent work from Daskalos et al (2011) has shown that the overexpression of p73, a well stablish cancerrelated gene associated with apoptosis and DNA repair (Zaika et al. 2011), might be associ‐

al. 2002). Furthermore, a growing body of evidence has shown that aberrant DNA methyla‐ tion (DNA hypermethylation or DNA hypomethlation) is an early event in carcinogenesis (Jones et al. 2002; Herman et al. 2003; Kopelovich et al. 2003). In addition, aberrant DNA methylation has been recently considered to be an excellent candidate to explain how cer‐ tain environmental factors may increase the risk of developing cancer (Chan et al. 2006). Ac‐ cordingly, an emerging catalog of cancer-related genes inactivated by DNA hypermethylation in gastric cancer has been established (Hamilton et al. 2006; Wu et al. 2006; Bernal et al. 2008). However, there are limited reports on DNA hypermethylation anal‐ ysis in precursor lesions of gastric cancer. Kang et al. (2001) tested five genes (p16, hMLH1, DAP-kinase, THBS1, and TIMP-3) in precancerous conditions to identify three different classes of hypermethylated genes. Hypermethylation of DAP-kinase was found in all pre‐ malignant conditions, whereas hMLH1 and p16 were preferentially methylated in intestinal metaplasia (6.3% and 2.1%, respectively), adenomas (9.8% and 11.5%, respectively) and gas‐ tric cancer (20.3% and 42.2%, respectively). THBS-1 and TIMP-3 were also hypermethylated at a similar frequency in all premalignant conditions, but showed a marked increase from chronic gastritis to intestinal metaplasia (10.1% & 34.7% and 14.5% & 36.7%, respectively; P < 0.05), as well as from adenomas to carcinomas (28.3% & 48.4% and 26.7% & 57.4% respec‐ tively). Another study including 11 genes (COX-2, DAP-kinase, E-cadherin, GSTP1, MGMT, hMLH1, p14, p16, THBS1, TIMP3, and RASSF1A) and 268 premalignant conditions (Kang et al. 2003) identified specific patterns of aberrant DNA methylation associated to aging. Spe‐ cifically five genes (DAP-kinase, E-cadherin, p14, THBS1, and TIMP-3) showed a progres‐ sive increase in aberrant methylation frequency as a function of aging, whereas the other genes (COX-2, GSTP1, MGMT, hMLH1, p16, and RASSF1A) were rarely methylated. Male patients showed higher numbers of hypermethylated genes than females (3.2 vs. 2.1, respec‐ tively, P = 0.002) and cases with severe intestinal metaplasia also showed a higher frequency of aberrantly methylated genes. Taken together, these findings suggest that hypermethyla‐ tion occurs early in the multistep gastric carcinogenesis and that accumulate during this process. In addition, age and gender are closely associated with increased frequency of aber‐ rant DNA methylation. A further study demonstrated that DNA methylation of E-cadherin was associated with H. pylori infection (p=0.002) in an independent way of age and/or gas‐ tritis (Chan et al. 2003). Similarly, Maekita et al. (2006) carried out a more detailed analysis of the effect of H. pylori infection in the progression of aberrant DNA methylation in chronic gastritis. These authors collected gastric mucosa samples from 154 healthy volunteers (56 H. pylori negative and 98 H. pylori positive) and from 72 cases with gastric cancers (29 H. pylo‐ ri negative and 43 H. pylori positive). Among the healthy volunteers, methylation levels were 5.4- to 303-fold higher in H. pylori positive subjects than in H. pylori negative ones (P < 0.0001). Particularly, methylation levels of the LOX, HAND1 and THBD at their promoter regions were identified in H. pylori-positive individuals. Among H. pylori-negative individ‐ uals, methylation levels were 2.2- to 32-fold higher in gastric cancer cases than in age-match‐ ed healthy volunteers. These findings suggest that H. pylori infection potently induces aberrant DNA methylation of several genes beyond E-cadherin, and that methylation levels of specific genes seemed to reflect the risk of gastric cancer in H. pylori-negative subjects. A recent quantitative analysis of the promoter region of a novel gene Reprimo (RPRM), per‐

84 Current Topics in Gastritis - 2012

ated with hypomethylation of the P2 promoter region that controls the DeltaNup73 isoform. Although this work was not performed in a gastric cancer model, our preliminary findings suggests similar results in gastric cancer cases (Corvalán et al. 2008). Based on both data, we decide to analysed the protein overexpression of p73 as well as other seven well stablished cancer-related genes activated by hypomethylation (BRCA1, HSP90, STAT1, FHIT, EGFR, p73, p53, p16INK4a) (Tanaka et al. 1998; Nien et al. 2007; Shutoh et al. 2009; Yu et al. 2009; Valdez et al. 2010; Daskalos et al. 2011; Rusiecki et al. 2011; Szaumkessel et al. 2011; Fornari et al. 2012). This analysis was performed in a large number of matched tumor/non-tumor adjacent mucosa of early gastric cancer as well as non-tumor cases at different stages of the multistep cascade of gastric cancer (Carrasco et al. 2010). Among 8 genes tested, only the overexpression of p73 was higher in matched tumor/non-tumor adjacent mucosa than in chronic gastritis/intestinal metaplasia (50.5% vs. 10.8%; P < 0.0001). We also assessed a detail histopathological analysis of the precursor lessions of gastric cancer through the Sydney and OLGA systems (Dixon et al. 1996; Corvalan et al. 2011; Rugge et al. 2011). As has been de‐ scribed (Rugge et al. 2010) only severe atrophy and OLGA stage IV were the most relevant features to identified high-risk gastritis (Fig. 4). However, the most interesting finding come after our attempt to integrated protein expression and histopathological assessments. This integration was performed by Significance Analysis of Microarrays (SAM), a multiple test‐ ing approach method that has been extensively applied in genomic research (Tusher et al. 2001) and confirmed by logistic regression (Nick et al. 2007). This integrative approach led us to identified that overexpression of p73 was even more significant than severe atrophy and OLGA stage IV in identifying high-risk premalignant gastritis (Carrasco et al. 2010). Therefore, we believe that the identification of the overexpression of p73 might contribute greatly to risk assessment of chronic gastritis to the development of gastric cancer.

**Figure 4.** Serial Analysis for Microarray from Non-tumor adjacent mucosa (NTAM) and chronic gastritis controls. NTAM group is significantly characterized by the overexpression of p73, OLGA stages III to IV, and severe atrophy (ATR-3), intestinal metaplasia (IM-3), and chronic inflammation (CI-3) according to the Sydney System. Control group cases were significantly characterized by lack of intestinal metaplasia (IM-0), atrophy (ATR-0), and chronic inflamma‐

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http://dx.doi.org/10.5772/53131

87

Data presented here suggests that genetic, epigenetic by means of DNA methylation might play a role in the dinamics of the progression of chronic gastritis and premalignant cascade to gastric cancer. DNA methylation works in both ways, inactivating or activating tumorrelated genes through the hypermethylation or hypomethylation of the promoter regions of specific genes. The integration of these molecular bases of chronic gastritis with histolopa‐ thological assessment by Sydney and OLGA systems will contribute to the better risk assess‐

Grant Support: Grants-in-Aid for Fondo Nacional de Ciencia y Tecnologia (FONDECYT) #1111014 and Fondo de Fomento al Desarrollo Científico y Tecnológico (FONDEF)

tion (CI-0). False discovery rate = 0. Taken from Carrasco et al., Clin.Cancer Res 2010;16:3253-9.

**4. Conclusion**

ment for the development of gastric cancer.

#D09i1137 to A.H. Corvalan from the Government of Chile.

**Acknowledgements**

**Figure 3.** The effect of the eradication of H. pylori in the pattern of DNA methylation as an example of environmental factors and the susceptibility of gastric cancer. (A) Before eradication of H. pylori (week 0), methylation was present in patients 3, 5 and 6. (B) Bisulfite sequencing confirm the methylated product. (C) After eradication of H. pylori (week 6), methylation was not present in any patient. (D) Bisulfite sequencing confirm the absence of methylated cytosine. MW: molecular weight marker, U: unmethylated band, M: methylated band, red color: unmethylated cytosines converted to thymidine, blue color: methylated cytosines. Taken from Chan et al., Gut 2006;55:463-8 with permission from BMJ Publishing Group Ltd.

**Figure 4.** Serial Analysis for Microarray from Non-tumor adjacent mucosa (NTAM) and chronic gastritis controls. NTAM group is significantly characterized by the overexpression of p73, OLGA stages III to IV, and severe atrophy (ATR-3), intestinal metaplasia (IM-3), and chronic inflammation (CI-3) according to the Sydney System. Control group cases were significantly characterized by lack of intestinal metaplasia (IM-0), atrophy (ATR-0), and chronic inflamma‐ tion (CI-0). False discovery rate = 0. Taken from Carrasco et al., Clin.Cancer Res 2010;16:3253-9.

#### **4. Conclusion**

ated with hypomethylation of the P2 promoter region that controls the DeltaNup73 isoform. Although this work was not performed in a gastric cancer model, our preliminary findings suggests similar results in gastric cancer cases (Corvalán et al. 2008). Based on both data, we decide to analysed the protein overexpression of p73 as well as other seven well stablished cancer-related genes activated by hypomethylation (BRCA1, HSP90, STAT1, FHIT, EGFR, p73, p53, p16INK4a) (Tanaka et al. 1998; Nien et al. 2007; Shutoh et al. 2009; Yu et al. 2009; Valdez et al. 2010; Daskalos et al. 2011; Rusiecki et al. 2011; Szaumkessel et al. 2011; Fornari et al. 2012). This analysis was performed in a large number of matched tumor/non-tumor adjacent mucosa of early gastric cancer as well as non-tumor cases at different stages of the multistep cascade of gastric cancer (Carrasco et al. 2010). Among 8 genes tested, only the overexpression of p73 was higher in matched tumor/non-tumor adjacent mucosa than in chronic gastritis/intestinal metaplasia (50.5% vs. 10.8%; P < 0.0001). We also assessed a detail histopathological analysis of the precursor lessions of gastric cancer through the Sydney and OLGA systems (Dixon et al. 1996; Corvalan et al. 2011; Rugge et al. 2011). As has been de‐ scribed (Rugge et al. 2010) only severe atrophy and OLGA stage IV were the most relevant features to identified high-risk gastritis (Fig. 4). However, the most interesting finding come after our attempt to integrated protein expression and histopathological assessments. This integration was performed by Significance Analysis of Microarrays (SAM), a multiple test‐ ing approach method that has been extensively applied in genomic research (Tusher et al. 2001) and confirmed by logistic regression (Nick et al. 2007). This integrative approach led us to identified that overexpression of p73 was even more significant than severe atrophy and OLGA stage IV in identifying high-risk premalignant gastritis (Carrasco et al. 2010). Therefore, we believe that the identification of the overexpression of p73 might contribute

greatly to risk assessment of chronic gastritis to the development of gastric cancer.

**Figure 3.** The effect of the eradication of H. pylori in the pattern of DNA methylation as an example of environmental factors and the susceptibility of gastric cancer. (A) Before eradication of H. pylori (week 0), methylation was present in patients 3, 5 and 6. (B) Bisulfite sequencing confirm the methylated product. (C) After eradication of H. pylori (week 6), methylation was not present in any patient. (D) Bisulfite sequencing confirm the absence of methylated cytosine. MW: molecular weight marker, U: unmethylated band, M: methylated band, red color: unmethylated cytosines converted to thymidine, blue color: methylated cytosines. Taken from Chan et al., Gut 2006;55:463-8 with permission from BMJ

Publishing Group Ltd.

86 Current Topics in Gastritis - 2012

Data presented here suggests that genetic, epigenetic by means of DNA methylation might play a role in the dinamics of the progression of chronic gastritis and premalignant cascade to gastric cancer. DNA methylation works in both ways, inactivating or activating tumorrelated genes through the hypermethylation or hypomethylation of the promoter regions of specific genes. The integration of these molecular bases of chronic gastritis with histolopa‐ thological assessment by Sydney and OLGA systems will contribute to the better risk assess‐ ment for the development of gastric cancer.

#### **Acknowledgements**

Grant Support: Grants-in-Aid for Fondo Nacional de Ciencia y Tecnologia (FONDECYT) #1111014 and Fondo de Fomento al Desarrollo Científico y Tecnológico (FONDEF) #D09i1137 to A.H. Corvalan from the Government of Chile.

#### **Author details**

Alejandro H. Corvalan, Gonzalo Carrasco and Kathleen Saavedra

Ponfiticia Universidad Catolica de Chile, Chile

#### **References**

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**Author details**

88 Current Topics in Gastritis - 2012

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Alejandro H. Corvalan, Gonzalo Carrasco and Kathleen Saavedra

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