What is the relationship between free-radical release and extent of mucosal damage?
G.J. Wetscher, C. Profanter (Innsbruck),
R.A. Hinder (Jacksonville)
Gastroesophageal reflux disease (GERD) is the most common foregut problem and may frequently lead to complications such as ulceration, stricture or Barrett's esophagus with an increased risk for the development of esophageal adenocarcinoma [1, 2].
The role of oxygen-derived free radicals in GERD has been evaluated in recent animal studies [3-5]. Oxygen-derived free radicals are produced in a chain reaction leading from the superoxide anion via hydrogen peroxide to hypochlorous acid and to the very toxic hydroxyl free radical which is produced in only small amounts . The main source of free radical production is the electron transfer of the respiratory chain of mitochondria [7, 8]. Oxygen-derived free radicals cause cell death by destroying membrane lipids . They may also lead to DNA modulation which is considered to be an important mechanism of carcinogenesis [10, 11]. Dismutation of superoxide anions by superoxide dismutase (SOD) which interrupts the free radical chain reaction at the very beginning of the reaction prevents reflux esophagus in rats  (Figures 1A and 1B). This demonstrates that oxygen-derived free radicals mediate reflux esophagitis in rats and it is mainly the superoxide anion which induces cell damage . Mixed reflux of gastric and duodenal juice causes significantly more free radical damage in the rat esophagus than pure acid reflux . This supports the role of duodenogastroesophageal reflux in complicated GERD as observed in clinical studies [1, 12]. In a recent study we evaluated the implication of oxygen-derived free radicals in reflux esophagitis of humans [13, 14]. In this study we also investigated whether free radical production correlates with the grade of esophagitis and whether Barrett's esophagus is a severe form of free radical damage.
Figure 1. A. Rat esophagus exposed to mixed reflux for 24 hours. Note the severe ulcerous esophagitis. B. Rat esophagus exposed to mixed reflux + SOD treatment. There is no evidence of macroscopic esophagitis. (Reproduced with permission from .) (See page XXIV for colour figure.)
Patients and methods
Multiple esophageal biopsies were taken from 5 cm above the gastroesophageal junction in 18 control patients and in 35 GERD patients with various grades of reflux esophagitis. Additional biopsies were taken from the lesser curvature 2 cm below the gastroesophageal junction in 5 of the reflux patients in order to evaluate whether free radical damage found in Barrett's esophagus is due to gastroesophageal reflux or due to the type of the epithelium. Three of these 5 patients had Barrett's esophagus with severe associated esophagitis.
Reflux esophagitis was endoscopically classified by means of the Savary-Miller classification system . Mild esophagitis was macroscopically defined as grade 0 to grade 2, and severe esophagitis as grade 3 to 4. Barrett's metaplasia was diagnosed when specialized epithelium was found in the biopsies .
Only patients with an incompetent lower esophageal sphincter (LES) were included in the study .
Production of oxygen-derived free radicals in the biopsies was measured by means of chemiluminescence . In addition we assessed the lipid peroxidation which is a marker of membrane damage caused by oxidative stress .
Results of the measurement chemiluminescence and of lipid peroxidation are shown in Figures 2A and 2B. Chemiluminescence and lipid peroxidation were significantly increased in GERD patients. Highest levels were found in the group with severe reflux esophagitis and in both groups with Barrett's esophagus. There was no significant difference between severe esophagitis, Barrett's esophagus with severe associated esophagitis or Barrett's esophagus with mild associated esophagitis. There was a positive correlation between the grade of esophagitis and chemiluminescence or lipid peroxidation (Figures 3A and 3B). Gastric biopsies showed significantly lower levels of chemiluminescence than esophageal biopsies of control subjects whereas there was no significant difference regarding lipid peroxidation.
Figure 2. A. Chemiluminescence (counts/mg protein/ml). B. Lipid peroxidation in esophageal biopsies of control subjects and patients with various grades of esophagitis and in gastric biopsies. Data are mean ± SEM. * p < 0.05 versus control, + p < 0.05 versus mild esophagitis.
Figure 3. Spearman regression analysis between the grade of esophagitis and chemiluminescence (A) or lipid peroxidation (B).
This study demonstrates that reflux esophagus in humans is mediated by oxygen-derived free radicals. The grade of esophagitis correlates positively with oxidative stress in the esophageal mucosa. Since highest levels of free radical production were found in patients with Barrett's esophagus it may be concluded that Barrett's esophagus is a severe form of GERD. The enhancement of free radical production in Barrett's esophagus is not due to the fact that this is a different type of epithelium, similar to gastric mucosa, since low levels of free radical damage were found in the gastric mucosa.
Oxygen-derived free radicals cause DNA damage [10, 11], thereby acting as carcinogens. Therefore our data may explain the increased risk of Barrett's esophagus for the development of Barrett's cancer [1, 2].
Barrett's esophagus may be associated with mild or severe esophagitis but there is no significant difference regarding free radical production. High levels of endogeneous SOD have been found in Barrett's mucosa associated with mild esophagitis and this may protect against severe esophagitis  since it is mainly the superoxide anion which causes reflux esophagitis . Whether high SOD levels in some patients with Barrett's esophagus may protect against cancer is doubtful. DNA damage is mainly caused by the hydroxyl free radical  and this can be produced in small amounts in a second pathway via the Fenton-type reaction despite blockage of the free radical chain reaction with SOD .
Since reflux esophagitis is meadiated by free radicals, effective therapy aimed at the inhibition of free radical production is required to prevent complications. It has been shown that this can be accomplished with antireflux surgery which reduces free radical production to control levels . Whether medical therapy aimed at suppression of gastric acid production is as successful as surgical therapy is doubtful since it does not prevent duodenogastroesophageal reflux, which is implicated in complicated GERD . Prospective studies are required to answer this question. The clinical use of free radical scavengers is limited. In animal studies only SOD has been shown to be effective in preventing reflux esophagitis . At present no SOD preparations exist with a reasonably long half-life which would be needed in humans.
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