Is the concentration of reactive oxygen species produced in Barrett's mucosa a contributor to malignant transformation?

A. Farhadi, J.Z. Fields, A. Keshavarzian (Chicago)

Gastroesophageal reflux disease (GERD) is one of the most important gastrointestinal diseases in the Western world because it is extremely common - over 30% of the general population suffers from at least monthly reflux episodes - and because it can lead to cancer [1-3].

It is now well-established that chronic GERD increases the risk of esophageal cancer - a cancer whose incidence has risen faster than any other malignancy in recent years in the United States, and now represents the most common histologic type of esophageal cancer observed in major institutions [4]. This trend is not only true for western countries [5, 6]; it is also true in countries such as China and Iran where adenocarcinoma of the esophagus used to be extremely rare and squamous cell carcinoma of the esophagus used to be common (personal communication). For example, the incidence of adenocarcinoma of the esophagus has tripled during the last 25 years. The most plausible reason for this astonishing increase is reflux.

It is also now known that GERD predisposes patients to adenocarcinoma of the esophagus only if it is associated with Barrett's epithelium [7]. It is now well-established that reflux esophagitis and transformation of squamous epithelium of the esophagus to columnar epithelium/intestinal metaplasia (i.e., Barrett's esophagus) is due to chronic and significant exposure of esophageal epithelium to gastric contents including acid, pepsin and or bile [7, 8]. However, the mechanism of tissue injury and how this injury predisposes the esophagus to malignant transformation is not known and cannot simply be attributed to acid exposure. For example, esophagitis and Barrett's epithelium can be absent in spite of significant gastroesophageal reflux of acid. Hence, other factors appear to be necessary for significant esophageal tissue damage (esophagitis), metaplasia of esophageal epithelium (Barrett's) and adenocarcinoma.

One likely candidate factor is the group of oxidants known as reactive oxygen species (ROS) a group of chemicals that has been implicated in numerous chronic disorders and aging. For example, the role of ROS has been well established in the pathogenesis of ischemic injury of the gastrointestinal mucosa, inflammatory bowel disease [9-12], experimental colitis [13], peptic ulcer disease [14, 15], and in other types of the mucosal damage induced by non-steroidal anti-inflammatory drugs [16], ethanol [17], haemorrhagic shock [18], feeding restriction stress [19], platelet activating factor [20] and Helicobacter pylori [21, 22].

ROS include oxygen metabolites that form by one or two electron reduction of oxygen such as superoxide anion (O2 -) and hydrogen peroxide (H2O2). Other endogenous ROS such as hypochlorous acid [9] and the highly reactive hydroxyl radical (OHo) are formed in the presence of transition metal ions such as Fe2+ [23]. ROS are also generated in the body as products or by-products of several enzymes catalyzed reactions during cellular metabolism. It is therefore not surprising that ROS are present in all cells including the gastrointestinal tract. Additionally, ROS can reach the tissues of the body from exogenous sources such as diet. This is particularly true for the upper gastrointestinal tract such as esophagus and stomach. Potential sources of ROS in the esophagus, particularly when it is inflamed (esophagitis), include activated inflammatory cells (e.g. neutrophils), the hypoxanthine-xanthine oxidase reaction, disrupted mitochondrial electron transport, metabolism of arachidonate via the lipoxygenase pathway, and vascular endothelial cells [24].

Therefore, it is reasonable to assume that ROS, if they are shown to be produced in excess in esophagitis, can contribute to esophageal tissue damage. This is based on the fact that (a) as stated above, the esophagus, and particularly the inflamed esophagus, can produce ROS, and (b) ROS have been implicated in tissue damage in a variety of inflammatory disorders of the gastrointestinal tract including IBD [9-12] and in malignancies including gastrointestinal malignancies such as colon cancer [25, 26]. Indeed, we demonstrated [27] that ROS levels, measured by chemiluminescence, are increased in esophageal tissue of patients with esophagitis and Barrett's epithelium. Subsequent studies [28, 29] confirmed our original finding and further demonstrated that there is a correlation between the severity of esophagitis and the level of ROS. The source of ROS appeared to be a combination of inflammatory cells as well as esophageal epithelial cells. For example, our data demonstrated that 30% to 45% of ROS in inflamed esophageal mucosa can be attributed to neutrophils. The mechanism of ROS mediated damage appears to be, at least in part, through lipid peroxidation of essential cellular membranes.

It is also plausible that ROS are not only involved in esophageal inflammation but also in esophageal carcinogenesis. For example, chronic inflammation is now recognized as a major risk factor for carcinogenesis in the gastrointestinal tract in general and in esophagus in particular [30]. Furthermore, ROS are known to be involved in other gastrointestinal cancers such as colon cancer [25, 26]. It is important to note that several tumor markers such as p53 mutation are involved in both colon cancer and in adenocarcinoma of the esophagus [7]. However, there is no direct evidence in man to link ROS with esophageal adenocarcinoma.

In contrast, there are several strong lines of experimental evidence in animal models that implicate ROS in both chronic esophageal inflammation, esophageal epithelial metaplasia (Barrett's) and esophageal adenocarcinoma. For example, several investigators have demonstrated the development of esophageal metaplasia and esophageal adenocarcinoma after surgically induced duodenoesophageal reflux [30-32]. In addition it has been demonstrated that ROS levels are increased in the inflamed esophagus and the inflamed esophagitis is commonly transformed to Barrett's epithelium and esophageal adenocarcinoma [33, 34]. Furthermore, they have shown that antioxidants prevent these transformations [33] while iron supplements worsen oxidative stress as well as esophagitis and esophageal adenocarcinoma [8, 35]. Additionally, tissue oxidative damage has been demonstrated in these tissues as indicated by high levels of proteins carbonyls (protein oxidation), thiobarbituric acid reactive substances (lipid oxidation), and 8-OH-2deoxyguanosine (DNA oxidation) [35]. These studies also demonstrated that columnar epithelium in the esophagus are the target cells of oxidative damage, supporting the notion that oxidative DNA damage is a major factor in transformation to malignancy of chronically inflamed esophageal epithelium [35].

Since the above chain of events demonstrated in this animal model also occurs in man, it is reasonable to conclude that ROS are also involved in esophageal carcinogenesis in patients with chronic esophageal inflammation and Barrett's epithelium. That is, excess production of ROS by inflammatory cells and irritated epithelial cells contributes to esophagitis and transformation of stem cells to columnar metaplasia and then intestinal metaplasia. DNA damage induced by ROS contributes to key sequential mutations that eventually result in dysplasia and adenocarcinoma. Further studies in man are needed to confirm this possibility and to evaluate the effectiveness of antioxidants in prevention and treatment of Barrett's esophagus and prevention of esophageal adenocarcinoma.

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