**Expression of Reactive Oxygen Species in Reflux Disease**

Emma Andreasson and Anna Casselbrant

*Department of Gastrosurgical Research and Education, Institute of Clinical Sciences, Sahlgrenska Academy, University of Gothenburg, Sweden* 

## **1. Introduction**

78 Gastrointestinal Endoscopy

Vasilopoulos, S., Murphy, P., & Auerbach, A. (2002). Gastrointest Endosc*. The small-caliber* 

Zuber-Jerger, I., Ratiu, N., & Kullman, F. (2006). J Gastrointestin Liver Dis. *Long-lasting effect* 

*esophagitis*, Vol.55, No.1, (January 2002), pp. 99-106, ISSN 0016-5107

No.2, (June 2006), pp. 167-170, ISSN 1842-1121

*esophagus: an unappreciated cause of dysphagia for solids in patients with eosinophilic* 

*of endoscopic dilation of an esophageal stenosis due to eosinophilic esophagitis*, Vol.15,

Reflux of acidic gastric contents, or bile and pancreatic enzymes into the esophagus may cause mucosal inflammation (esophagitis or red streak). This disorder is commonly called gastroesohageal reflux disease (GERD), and in signs of esophageal mucosal injuries the disorder is called 'erosive reflux disease' (ERD). If the reflux is frequent and long standing such episodes can elicit severe inflammation or damage of the esophageal squamous epithelium (1-2).

Oxygen is a requirement for life but oxygen metabolites can cause serious tissue injuries. It is normal for the immune system to respond to injury to the mucosa or pathogens by producing oxygen and nitrogen radicals. Reactive oxygen species (ROS) are an often-used term that includes true radicals that have unpaired electrons as well as chemicals that can gain or loose electrons. Oxidative stress is a general term used to describe the steady state of oxidative damage in a cell, tissue or organ, caused by ROS. If there is an unbalance between the production of ROS and the systems ability to detoxify the reactive species or easily repair the resulting damage, oxidative stress is caused and this is a reality in most living organisms. ROS are used in immune system to attack and eliminate pathogens but ROS are also involved in the development of many diseases such as atherosclerosis and cancer (3-5).

There are many different sources by which ROS are generated. Among a lot of enzymes and molecules that cause oxidative stress there are three major enzymes; myeloperoxidase (MPO), nicotinamide adenine dinucleotide phosphate oxidase (NADPH oxidase) and nitric oxide syntase (NOS) that produce the products superoxide (O2 - ), hydrogen peroxide (H2O2), nitric oxide (NO) and hypocloric acid (HOCl).

We have previously shown that extremely high levels of NO are formed when nitrite in swallowed saliva meets acidic refluxates in the distal esophagus (6). NO has radicalcharacteristics and has been suggested to initiate esophageal carcinogenesis (7). This view may be questioned because luminally formed NO appears to be quite harmless and is rapidly eliminated during normal tissue conditions. However, in inflamed tissue with production of ROS it is reasonable to assume that luminal NO will react with particularly O2- and form the extremely labile oxidising compound peroxynitrite (ONOO-). Such oxidative species have potential roles in all steps of carcinogenesis including DNA

Expression of Reactive Oxygen Species in Reflux Disease 81

substrate to identify immunoreactive proteins by means of chemiluminescense. Images were captured by a Chemidox XRS cooled CCD camera, and analyzed with Quantity One software (BioRad laboratories, Hercules, CA, USA). Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) antibody (Imgenex, San Diego, CA, USA) was used as control for equal loading, and for each tested sample the ratio of primarily antibody/GAPDH was

*Target protein Primary antibody Positive control* 

Hl-60 sc-2209 Santa Cruz

Nitrotyrosine 12-354 Upstate

RAW 264.7 sc-2212 Santa Cruz

Loading control for western blot

Serum was used as positive control

Serum was used as positive control

Hl-60 sc-2209 Santa Cruz

Upstate/Millipore

Upstate/Millipore

TransductionLab/BioSite

MPO; myeloperoxidase, iNOS; inducible nitric oxide synthase, NADPH-oxidase; nicotinamide adenine dinucleotide phosphate oxidase, GAPDH; Glyceraldehyde-3-phosphate dehydrogenase, IL; interleukin Table 1. Antibodies and controls used in Western Blot analyses and immunohistochemistry

The mucosal specimens were fixed in buffered 4% formaldehyde and embedded in paraffin. Sections for immunohistochemistry (3µm) were deparaffinized and then boiled for 15 min in 10mM citrate buffer (pH 6.0) for antigen retrieval. The Immunocruz TM Staining System (Santa Cruz Biotechnology, Santa Cruz, CA, USA) was used for the immunohistochemistry protocol. After inhibition of endogenous peroxidase activity, the slides were pre-incubated with serum block and then incubated with primary antibodies against MPO, NADPH oxidase iNOS and nitrotyrosine (see table 1) over night in dilutions of 1:200, 1:50, 1:1000 and

**MPO** Antimyeloperoxidase 07-496

06-284

N 32030

H-195 sc-14015 Santa Cruz

phosphate dehydrogenase Imgenex/BioSite

Sc-52012 Santa Cruz

Sc-28343 Santa Cruz

**Nitrotyrosine** Antinitrotyrosine

**GAPDH** Glyceraldehyde-3-

**IL-1**β Interleukin-1 Beta

**IL-6** Interleukin-6

**iNOS** Transinos

**NADPH-oxidase** 

(p47phox)

**2.3 Immunohistochemistry** 

used.

mutation, activation of proto-oncogenes and inactivation or loss tumor suppressor genes (5, 7).

The aim of the present study is to elucidate the presence of radical producing enzymes, represented by NADPH oxidase, MPO and iNOS and also the expression of radical formation marker for ONOO- activity as well as nitro radical end products; nitrotyrosine. The second aim is to elucidate the histological changes and number of inflammatory cells as well as the expression of inflammatory markers IL1β and IL6 in human esophageal biopsies from healthy volunteers and patients with ERD.
