**4.2 Histological signs of inflammation**

No signs of active inflammation, defined as presence of lymphocytes, amount of eosinophiles granulocyts or plasmacells, were detected in the esophageal mucosa (table 3).

Expression of Reactive Oxygen Species in Reflux Disease 85

Fig. 3. Immunostaining of the human esophageal epithelium at X40 magnification with brown indicating positive immunoreactivity. (Panel A) Staining for MPO was localized in lymphocytes (arrow) as well as in upper layer epithelial cells. (Panel B) Immunostaining for NADPH oxidas is most obvious in basal epithelial cells and around the papillae (see arrows). (Panel C) Staining for iNOS was localized in stratum superficiale and spinosum (arrow). (Panel D) Immunoreactivity for nitrotyrosine is most obvious in upper layer epithelial cells

Histological signs of erosive mucosal disease were confirmed. In the red streak, the squamous epithelium showed a significantly thicker basal cell layer (p=0.016), longer papillae (p=0.001), thicker total epithelium thickness (p=0.016) and wider intercellular space (p=0.003) compared to biopsies taken in healthy control subjects (Figure 4A-D). Furthermore, also when compared to unaffected squamous mucosa in ERD-patients the epithelium showed a significantly thicker basal cell layer (p=0.022), longer papillae (p=0.022), and thicker total epithelium thickness (p=0.001), compared to biopsies from

A B

C D

and around the papillae

healthy controls (Figure 4A-D).

**4.3 Histology** 

Fig. 2. Western blot analysis of the MPO (Panel A), NADPH oxidas (Panel B), iNOS (Panel C) and nitrotyrosine (Panel D) in human esophageal biopsies taken from macroscopically normal squamous mucosa in healthy subjects (n=7), patients with erosive reflux disease (ERD)(n=13) and from the red streak areas in ERD-patients (n=13). Tissue sample were taken in the 3 o´clock position 2 cm proximal to the gastro esophageal junction. Significant differences are indicated with asterisks (\*=p≤0.01; Mann-Whitney U-test). The median value in each group is indicated


ERD; Errosive reflux disease

Table 3. The number of inflammatory cells in human esophageal mucosa

Optical density for NADPH oxidase

Optical density for nytrotyrosine

3

2.5

2

1.5

1

0.5

0

A B C A B C

10

8

6

4

2

0

A B C A B C A. Control; B. ERD; C. Red streak Fig. 2. Western blot analysis of the MPO (Panel A), NADPH oxidas (Panel B), iNOS (Panel C) and nitrotyrosine (Panel D) in human esophageal biopsies taken from macroscopically normal squamous mucosa in healthy subjects (n=7), patients with erosive reflux disease (ERD)(n=13) and from the red streak areas in ERD-patients (n=13). Tissue sample were taken in the 3 o´clock position 2 cm proximal to the gastro esophageal junction. Significant differences are indicated with asterisks (\*=p≤0.01; Mann-Whitney U-test). The median value in each group is indicated

 **Control ERD Red streak** 

**Mucosal lymphocytes** 6.29±2.0 5.8±0.9 8.9±2.1 **Eosinophilic granulocytes** 0 4.6±3.1 0.67±0.4

**Plasma cells** 0 0 0

Table 3. The number of inflammatory cells in human esophageal mucosa

**A B**

Optical density for MPO

Optical density for iNOS

1

0.8

0.6

0.4

0.2

0

0.5

0.4

0.3

0.2

0.1

0

ERD; Errosive reflux disease

**C D**

Fig. 3. Immunostaining of the human esophageal epithelium at X40 magnification with brown indicating positive immunoreactivity. (Panel A) Staining for MPO was localized in lymphocytes (arrow) as well as in upper layer epithelial cells. (Panel B) Immunostaining for NADPH oxidas is most obvious in basal epithelial cells and around the papillae (see arrows). (Panel C) Staining for iNOS was localized in stratum superficiale and spinosum (arrow). (Panel D) Immunoreactivity for nitrotyrosine is most obvious in upper layer epithelial cells and around the papillae

#### **4.3 Histology**

Histological signs of erosive mucosal disease were confirmed. In the red streak, the squamous epithelium showed a significantly thicker basal cell layer (p=0.016), longer papillae (p=0.001), thicker total epithelium thickness (p=0.016) and wider intercellular space (p=0.003) compared to biopsies taken in healthy control subjects (Figure 4A-D). Furthermore, also when compared to unaffected squamous mucosa in ERD-patients the epithelium showed a significantly thicker basal cell layer (p=0.022), longer papillae (p=0.022), and thicker total epithelium thickness (p=0.001), compared to biopsies from healthy controls (Figure 4A-D).

Expression of Reactive Oxygen Species in Reflux Disease 87

Spearman's r=0.65, p<0.05 Spearman's r=0.73, p<0.05

Optical density for Nitrotyrosine

 3 2.5 2 1.5 1 0.5 0 -0.5  3 2.5 2 1.5 1 0.5 0 -0.5

Optical density for Nitrotyrosine

Length of papillae Total epithelium thickness

100 200 300 400 500 200 300 400 500 600 700 800

esophageal mucosa a number of reactive changes were observed. Firstly, the histological signs of erosive mucosal diseases were confirmed, also in macroscopically normal squamous epithelium from ERD-patients were changed. Secondly, there were clear signs of increased radical forming capacity in the epithelium despite absence of histological inflammation. Taken together these findings indicate that a significant change of the esophageal mucosa

Nitration of tyrosine results in nitrotyrosine. The nitrate is mainly donated by peroxynitrite, however, nitrotyrosine formation is not solely generated by ONOO-. NADPH oxidase is a transmembrane electron transport chain, and the active NADPH oxidase catalyzes the production of O2- that serves as starting material for the production of different ROS (12). Oxidation of arginine by NOS creates the gas NO. NO reacts rapidly with O2- to produce the extremely reactive radical ONOO- which also can protonate and dissociate to give nitrogen (NO2) (5). MPO reacts with H2O2 formed by the respiratory burst to generate HOCl that further can form nitryl chloride (NO2Cl) (13). NO2 and NO2Cl can then nitrogenate tyrosine to form nitrotyrosine. It follows that all enzymes used in the study; MPO, NADPH oxidase,

Nitrotyrosine is a very stable altered amino acid (an addition of a nitro group to the benzene ring of tyrosine) that can be found both as a single amino acid and belong to a complete protein in the cell. It has been found in elevated levels in a lot of inflammatory conditions like atherosclerosis, rheumatoid arthritis, influenza, pancratitis, cholecystitis, Diabetes Mellitus, ulcerative colitis and Crohn´s disease (5, 14-15) and is therefore a good indicator of the radical production. In the present study nitrotyrosine showed elevated levels in both the red streak and in the squamous epithelium taken from the ERD-patients compared to controls. A number of studies have shown that MPO is elevated in reflux esophagitis, Barrett´s esophagus and adenocarcinoma (16-17). Eero *et al.* suggest that MPO is a key component of the pathway leading to oxidative stress and damage in the esophageal mucosa. They shown a step-by-step increase in MPO activity related to the severity of reflux disease (17). The expression of MPO was significant increased in the present study in both the red streak and

(A) (B)

and iNOS activity may be involved in the formation of nitrotyrosine.

area from patients with erosive reflux disease

had occurred in association to ERD.

Fig. 5. Positive correlation of nitrotyrosine expression with histopathological changes papillary length (A) and total epithelium thickness (B) in biopsies taken in the red streak

Fig. 4. Histological appearance in biopsies from macroscopically normal squamous mucosa of control subjects and patients with erosive reflux disease (ERD), and from the red streak areas in ERD-patients. Tissue sample were taken in the 3 o´clock position 2 cm proximal to the gastro esophageal junction. (Panel A) thickness of basal cellular layer, (Panel B) length of intraepithelial papillae, (Panel C) total epithelial thickness, and (Panel D) dilatation of intracellular space. Significant differences are indicated with asterisks (\*=p≤0.01; Mann-Whitney U-test, #=p<0.01; Wilcoxon´s signed rank test). Data is showed as means±SEM

#### **4.4 ROS expression correlation analysis with histopathological alterations and immunocells**

Correlation analysis revealed a positive correlation of increased expressions of nitrotyrosine with the histopathological alteration PL (r=0.65, p=0.05) and total epithelium thickness (r=0.73, p=0.025) in biopsies taken in the red streak area from ERD-patients (Figure 5A, B). No other correlation was found nor for the histopathological alterations or the number of inflammatory cells (data not shown).

### **5. Discussion**

This study is attempted to elucidate the total generation of ROS that is produced by enzymes and molecules in the esophageal mucosa. In the present exploration of the human

400

∗ ∗ ∗ ∗

300

200

Length of papillae (μm)

100

0

5

∗ ∗ ∗ ∗

#

A B C

4

3

Dilatation of intercellular space

A B C A B C

A. Control; B. ERD; C. Red streak Fig. 4. Histological appearance in biopsies from macroscopically normal squamous mucosa of control subjects and patients with erosive reflux disease (ERD), and from the red streak areas in ERD-patients. Tissue sample were taken in the 3 o´clock position 2 cm proximal to the gastro esophageal junction. (Panel A) thickness of basal cellular layer, (Panel B) length of intraepithelial papillae, (Panel C) total epithelial thickness, and (Panel D) dilatation of intracellular space. Significant differences are indicated with asterisks (\*=p≤0.01; Mann-Whitney U-test, #=p<0.01; Wilcoxon´s signed rank test). Data is showed as means±SEM

**4.4 ROS expression correlation analysis with histopathological alterations and** 

Correlation analysis revealed a positive correlation of increased expressions of nitrotyrosine with the histopathological alteration PL (r=0.65, p=0.05) and total epithelium thickness (r=0.73, p=0.025) in biopsies taken in the red streak area from ERD-patients (Figure 5A, B). No other correlation was found nor for the histopathological alterations or the number of

This study is attempted to elucidate the total generation of ROS that is produced by enzymes and molecules in the esophageal mucosa. In the present exploration of the human

2

(Arbitrary units)

1

0

**A B**

80

60

40

Basal cellular thickness (μm)

20

0

Total epithelial thickness (μm)

**immunocells** 

**5. Discussion** 

inflammatory cells (data not shown).

**C D**

A B C

Fig. 5. Positive correlation of nitrotyrosine expression with histopathological changes papillary length (A) and total epithelium thickness (B) in biopsies taken in the red streak area from patients with erosive reflux disease

esophageal mucosa a number of reactive changes were observed. Firstly, the histological signs of erosive mucosal diseases were confirmed, also in macroscopically normal squamous epithelium from ERD-patients were changed. Secondly, there were clear signs of increased radical forming capacity in the epithelium despite absence of histological inflammation. Taken together these findings indicate that a significant change of the esophageal mucosa had occurred in association to ERD.

Nitration of tyrosine results in nitrotyrosine. The nitrate is mainly donated by peroxynitrite, however, nitrotyrosine formation is not solely generated by ONOO-. NADPH oxidase is a transmembrane electron transport chain, and the active NADPH oxidase catalyzes the production of O2- that serves as starting material for the production of different ROS (12). Oxidation of arginine by NOS creates the gas NO. NO reacts rapidly with O2 to produce the extremely reactive radical ONOO- which also can protonate and dissociate to give nitrogen (NO2) (5). MPO reacts with H2O2 formed by the respiratory burst to generate HOCl that further can form nitryl chloride (NO2Cl) (13). NO2 and NO2Cl can then nitrogenate tyrosine to form nitrotyrosine. It follows that all enzymes used in the study; MPO, NADPH oxidase, and iNOS activity may be involved in the formation of nitrotyrosine.

Nitrotyrosine is a very stable altered amino acid (an addition of a nitro group to the benzene ring of tyrosine) that can be found both as a single amino acid and belong to a complete protein in the cell. It has been found in elevated levels in a lot of inflammatory conditions like atherosclerosis, rheumatoid arthritis, influenza, pancratitis, cholecystitis, Diabetes Mellitus, ulcerative colitis and Crohn´s disease (5, 14-15) and is therefore a good indicator of the radical production. In the present study nitrotyrosine showed elevated levels in both the red streak and in the squamous epithelium taken from the ERD-patients compared to controls. A number of studies have shown that MPO is elevated in reflux esophagitis, Barrett´s esophagus and adenocarcinoma (16-17). Eero *et al.* suggest that MPO is a key component of the pathway leading to oxidative stress and damage in the esophageal mucosa. They shown a step-by-step increase in MPO activity related to the severity of reflux disease (17). The expression of MPO was significant increased in the present study in both the red streak and

Expression of Reactive Oxygen Species in Reflux Disease 89

compared healthy subjects. Moreover, in this study the parametric dilatation of intracellular space was significantly increased at the "red streaks" of the distal esophagus of ERDpatients compared to biopsies from adjacent normal-locking epithelium. The above findings confirm previous observations for biopsies taken in the red streak and in 3 o´clock position 2 cm proximal to the gastro esophageal junction (27). Strong support for the involvement of nitrotyrosine in the pathogenesis of mucosal abnormalities in the red streak was obtained

In conclusion, there were clear signs of increased radical forming capacity in the epithelium despite absence of histological inflammation, in association to ERD. The histomorphological changes in ERD associating with nitrotyrosine expression, thus mirroring the foregoing radical formation, may suggest a role in the pathogenesis of esophagus. During acidified refluxes, epithelial ROS production in combination with luminal NO formation, may

[1] Lundell LR, Dent J, Benett JR, Blum AL, Armstrong D, Galmiche JP, et al. Endoscopic

[2] Dent J, El-Serag HB, Wallander MA, Johansson S. Epidemiology of gastro-oesophageal

[3] Pilar J, Piazuelo E, Sánchez MT, Ortego J, Soteras F, Lanas A. Free radicals and

[4] Grisham M.B. Serial Review: Reactive oxygen species in immune responses. Free radical

[5] Grisham M.B Jourd´heuil D, Wink DA. Chronic inflammation and reactive oxygen and

[6] Casselbrant A, Pettersson A, Ruth M, Bove M, Lundell L, Fändriks L. Sources of intra-

[7] Suzuki H, Iijima K, Scobie G, Fyfe V, McColl KE. Nitrate and nitrosative chemistry within Barrett´s oesophagus during acid reflux. Gut 2005; 54:1527-35 [8] Edebo A, Vieth M, Tam W, Bruno M, van Berkel AM, Stolte M, et al. Circumferential and

[9] Bradford MM. A rapid and sensitive method for the quantitation of microgram

[10] Rieder F, Cheng L, Harnett KM, Chak A, Cooper GS, Isenberg G, Ray M, Katz JA,

assessment of oesophagitis: clinical and functional correlates and further validation

antioxidant systems in reflux esophagitis and Barrett´s esophagus. World J

nitrogen metabolism-implications in DNA damage and mutagenesis. Aliment

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axial distribution of esophagus mucosal damage in reflux disease. Dis Esophagus

quantities of protein utilizing the principle of protein-dye binding. Anal Biochem.

Catanzaro A, O´shea R, Post AB, Wong R, Sivak MV, Mccormick T, Phillips M, West GA, Wills JE, Biancani P, Fiocchi C. Gastroesophageal reflux disease-

This study was supported by an unconditional scientific grant from AstraZeneca.

of the Los Angeles classification. Gut 1999; 45:172-80

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**6. Acknowledgements** 

**7. References** 

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in the squamous epithelium from ERD-patients, however no correlation was found between MPO and the morphological changes.

The expression of NADPH oxidase was not increased in the ERD-patients compared to the healthy individuals. This could be compared with studies made on gastritis caused by Helicobacter Pylori where NADPH oxidase was significant higher in infected patients compared to healthy volunteers (18). Our finding may perhaps suggest the possibility that the reflux of acid is not strong enough as triggering, compared to pathogens. A recent study made by Feagins LA et al. have found that different components of gastric juice, acidic media or acidic bile acid media, induce ROS production through different mechanisms (19). Moreover, no signs of active inflammation were detected in the esophageal mucosa, defined as presence of lymphocytes, amount of eosinophiles granulocyts or plasmacells. The present study is also made on patients with mild esophagitis, classified to LA-A by the endoscopist (1). Several studies have looked at pro-inflammatory cytokines expression along the inflammation- metaplasia- dysplasia- adenocarcinoma sequence in the esophagus and have also found a stepwise-elevated expression correlate to grade of severity of the disease (20- 21). Such association was not made in the present study for IL-1β and IL-6 at protein level, whereas gene transcript for IL-6 was increased in ERD-patients indicating somewhat small alteration may exist beyond the detections level for protein.

We have previously shown that two sources of NO formation exist in esophagus, both dependent on the presence of acid in the esophageal lumen; enzymatic degradation of Larginine by NO synthase and non-enzymatic NO-production their nitrite from the salvia is reduced when it meets the extremely low pH in refluxate, a mechanism related to dietary intake of nitrate (6). The sources of body nitrate are intake through drinking water and vegetables, and endogenous synthesis (22). Vegetables vary greatly in their nitrate content, and water nitrate content also varies with geographical location (22). It follows that luminal NO formations differ between individual dependent on nitrate intake during acidic reflux.

Enzymatic NO formation is constantly expressed but may be activated upon presence of acid in esophageal lumen (6). Several studies have described the expression of iNOS in esophageal squamous epithelium which have been associated with pathological condition such as cell transformation but also suggested a function related to epithelial integrity (6, 23- 24). Thus, our results confirm the expression of iNOS in the surface epithelium. However, neither the biopsies taken from ERD-patients nor the red streak areas in ERD-patients were significant different in iNOS expression compared to controls.

The topographical organisation of the iNOS in the epithelium in combination with luminal non-enzymatic NO, may create particular conditions for NO gradients through the mucosa. Immunoreactivily to MPO and NADPH oxidase was also found in the surface epithelium. Therefore we could assume that during gastric acidic reflux huge level of NO is formed simultaneous with production of epithelial mucosal ROS leading to increased formation of ONOO-, which may contribute to cellular injury and DNA damage (7, 25).

MPO, NADPH oxidase and iNOS are usually found in phagocytes including neutrophils, eosinophils, monocytes and macrophages with the primary function of phagocytosis and destruction of microorganisms (26). However, except MPO that was localized in both lymphocytes and epithelial cells, immunostaining for NADPH oxidase and iNOS was *only* found in the esophageal epithelial cells. Thus, the presently increased radical forming capacity in the epithelium is independent of inflammatory cells.

In the morphological investigation of reflux signs we found that all parametric used, dilatation of intracellular spaces, papillary length, basal cell layer thickness, and total epithelium thickness were significantly increased in the distal esophagus in ERD-patients compared healthy subjects. Moreover, in this study the parametric dilatation of intracellular space was significantly increased at the "red streaks" of the distal esophagus of ERDpatients compared to biopsies from adjacent normal-locking epithelium. The above findings confirm previous observations for biopsies taken in the red streak and in 3 o´clock position 2 cm proximal to the gastro esophageal junction (27). Strong support for the involvement of nitrotyrosine in the pathogenesis of mucosal abnormalities in the red streak was obtained using correlation analysis.

In conclusion, there were clear signs of increased radical forming capacity in the epithelium despite absence of histological inflammation, in association to ERD. The histomorphological changes in ERD associating with nitrotyrosine expression, thus mirroring the foregoing radical formation, may suggest a role in the pathogenesis of esophagus. During acidified refluxes, epithelial ROS production in combination with luminal NO formation, may constitute aggravated factors in the carcinogenic process.
