NRF2 antioxidant response protects against acidic bile salts-induced oxidative stress and DNA damage in esophageal cells.

Gastroesophageal reflux disease (GERD) is the main risk factor for Barrett's tumorigenesis. In this study, we investigated the role of NRF2 in response to exposure to acidic bile salts (ABS), in conditions that mimic GERD, using Barrett's esophagus cell models. We detected an increase in NRF2 protein levels, following exposure to ABS. We found oxidization of cysteines (cysteines with oxidized thiol groups) in KEAP1 protein with a weaker interaction between NRF2 and KEAP1, following ABS exposure. Treatment with bile salts increased nuclear NRF2 levels, enhancing its transcription activity, as measured by an ARE (antioxidant response element) luciferase reporter assay. The mRNA expression levels of NRF2 target genes, HO-1 and GR, were increased in response to ABS exposure. Using genetic overexpression and knockdown of NRF2, we found that NRF2 has a critical role in suppressing ABS-induced ROS levels, oxidative DNA damage, DNA double strand breaks, and apoptosis. Collectively, our results suggest that transient induction of NRF2 in response to ABS plays a pivotal role in protecting esophageal cells by maintaining the levels of oxidative stress and DNA damage below lethal levels under GERD conditions.

Do Statins Increase the Risk of Esophageal Conditions? Findings from Four Propensity Score-Matched Analyses.

BACKGROUND AND OBJECTIVE: Statins are commonly used medications. Whereas some observational studies suggested an association of statin use with Barrett's esophagus and some upper gastrointestinal symptoms, there is a dearth of data on the association of statins and common esophageal conditions such as gastroesophageal reflux disease and esophagitis. The aim of this study is to examine the association of statins with esophageal conditions. METHODS: This is a retrospective cohort study using regional military healthcare data (1 October, 2003 to 1 March, 2012). The primary analyses evaluated the odds of: esophagitis; symptoms of esophagitis; gastroesophageal reflux disease/dyspepsia; and esophageal complications of gastroesophageal reflux disease in four propensity score-matched cohorts of statin users and non-users (propensity score-overall, propensity score-healthy, propensity score-women, and propensity score-men cohorts). Secondary and sensitivity analyses were performed. RESULTS: In the propensity score-overall cohort (n = 12,684), statin users were more likely to be diagnosed with esophagitis (odds ratio 1.11, 95% confidence interval 1.01-1.22) and gastroesophageal reflux disease/dyspepsia (odds ratio 1.18, 95% confidence interval 1.10-1.27) compared with non-users. Similar findings were seen in the propensity score-healthy cohort and in the propensity score-men cohort. In the propensity score-women cohort, the odds of esophagitis was higher among statin users compared with non-users (odds ratio 1.16, 95% confidence interval 1.02-1.32) but other outcomes were not different. In sensitivity analyses, which excluded patients with obesity, statin use was not associated with an increased odds ratio of gastroesophageal reflux disease/dyspepsia. CONCLUSION: Statin therapy was associated with higher odds of being diagnosed with esophagitis and gastroesophageal reflux disease/dyspepsia. Further study is warranted to elucidate the potential role of statins in these commonly diagnosed esophageal conditions.

Deoxycholic acid (DCA) confers an intestinal phenotype on esophageal squamous epithelium via induction of the stemness-associated reprogramming factors OCT4 and SOX2.

Barrett's esophagus (BE) is essentially a metaplasia in which the normal stratified squamous epithelium is replaced by columnar epithelium. This study focuses on the involvement of OCT4 and SOX2, 2 key cell-reprogramming factors, in the deoxycholic acid (DCA)-induced expression of the intestinal hallmarks Cdx2 and MUC2 using both in vivo and in vitro models. Up-regulated expression of OCT4 and down-regulated expression of SOX2 were observed in BE compared with normal esophagus and esophagitis. Consistent with the data in vivo, DCA induced time-dependent expression of OCT4 at both the mRNA and protein levels and decreased nuclear expression of SOX2 in Het-1A cells. Down-regulation of OCT4 expression by siRNA abrogated DCA-induced expression of Cdx2 and MUC2, whereas siRNA against SOX2 significantly upregulated the expression of both Cdx2 and MUC2. Our data indicate that both OCT4 and SOX2 play important roles in the development of BE triggered by bile acid reflux.

Bile acids but not acidic acids induce Barrett's esophagus.

Barrett's esophagus (BE) is associated with the development of esophageal adenocarcinoma (EAC). Bile acids (BAs) refluxing into the esophagus contribute to esophageal injury, which results in BE and subsequent EAC. We developed two animal models to test the role of BAs in the pathogenesis of BE. We surgically generated BA reflux, with or without gastric acid, in rats. In a second experiment, we fed animals separately with BAs and gastric acid. Pathologic changes were examined and the expression of Muc2 and Cdx2 in BE tissue was tested by immunostaining. Inflammatory factors in the plasma, as well as differentiation genes in BE were examined through highly sensitive ELISA and semi-quantitative RT-PCR techniques. We found that BAs are sufficient for the induction of esophagitis and Barrett's-like metaplasia in the esophagus. Overexpression of inflammatory cells, IL-6, and TNF-α was observed both in animals fed with BAs and surgically generated BA reflux. Furthermore, elevated levels of Cdx2, Muc2, Bmp4, Kit19, and Tff2 (differentiation genes in BE) were found in BA-treated rats. In conclusion, BAs, but not gastric acid, are a major causative factor for BE. We confirmed that BAs contribute to the development of BE by inducing the inflammatory response in the esophagus. Inhibiting BAs may be a promising therapy for BE.

Oral bisphosphonates and upper gastrointestinal toxicity: a study of cancer and early signals of esophageal injury.

SUMMARY: We evaluated the association between bisphosphonate use and (1) upper gastrointestinal cancer, (2) upper endoscopy, (3) incident Barrett's esophagus, and (4) prescription antacid initiation among Medicare beneficiaries. We found no bisphosphonate-cancer association and negative bisphosphonate-Barrett's association. INTRODUCTION: Bisphosphonates can irritate the esophagus; a cancer association has been suggested. Widespread bisphosphonates use compels continued investigation of upper gastrointestinal toxicity. METHODS: Using a 40% Medicare random sample denominator, inpatient, outpatient (2003-2011), and prescription (2006-2011) claims, we studied patients age 68 and older with osteoporosis and/or oral bisphosphonate use. Inverse propensity weighting estimated marginal structural models for the effect of bisphosphonate intensity (pills per month) and cumulative bisphosphonate pills received on upper gastrointestinal cancer risk. Secondary analyses of sub-cohorts without past bisphosphonates or upper endoscopy assessed bisphosphonate initiation and risk of (1) upper endoscopy, (2) incident Barrett's esophagus, and (3) prescription antacid initiation. RESULTS: The cohort included 1.64 million beneficiaries: 87.9% women, mean age, 76.8 (standard deviation (SD) 9.3); mean follow-up, 39.6 months; 38.1% received oral bisphosphonates. Cumulative bisphosphonate receipt, among users, ranged from 4 to 252 pills (5th to 95th percentile). We identified 2,308 upper gastrointestinal cancers (0.43/1000 person years). We found no association between cumulative bisphosphonate pills and cancer, odds ratio (OR) for each additional pill 1.00 (95% confidence interval (CI) 1.00, 1.00). In sub-cohorts, compared to none, lowest cumulative bisphosphonate use (one to nine pills) was associated with higher risk of endoscopy (OR 1.11, 95% CI 1.08-1.14) and antacid initiation (OR 1.13, 95% CI 1.10-1.16); higher intensity conferred no increased risk. Higher intensity and higher cumulative bisphosphonate category were associated with lower Barrett's risk. CONCLUSIONS: We found no bisphosphonate-cancer association and negative bisphosphonate-Barrett's association. Bisphosphonate initiation appears to identify patients susceptible to early irritating effects; clinicians might offer alternatives and delay endoscopy or antacids.

The use of nonsteroidal anti-inflammatory drugs and the risk of Barrett's oesophagus.

BACKGROUND: Epidemiological studies have consistently reported inverse associations between nonsteroidal anti-inflammatory drugs (NSAIDs) and oesophageal adenocarcinoma, but few have investigated associations with the precursor lesion, Barrett's oesophagus. AIM: To investigate the relationship between NSAID use and risk of Barrett's oesophagus. METHODS: We conducted a large population-based case-control study that collected information on patterns of intake for aspirin and non-aspirin NSAIDs during the past 5 years and other exposures from 285 patients with nondysplastic Barrett's oesophagus, 108 patients with dysplastic Barrett's oesophagus, and two separate control groups: 313 endoscopy patients with acute inflammatory changes ('inflammation controls') and 644 population controls. We calculated odds ratios (ORs) and 95% CIs using unconditional logistic regression. RESULTS: Use of aspirin was not associated with nondysplastic Barrett's oesophagus when compared with population (OR=1.01, 95% CI 0.71-1.43) or inflammation controls (OR=1.16, 95% CI 0.80-1.68). Whereas we observed significant risk reductions for use of non-aspirin NSAIDs when nondysplastic Barrett's oesophagus cases were compared with population controls (OR=0.69, 95% CI 0.49-0.97), the effect was weaker and nonsignificant when cases were compared with inflammation controls (OR=0.82, 95% CI 0.57-1.18), and no dose-response effects were present in either analysis. We found no evidence that aspirin or non-aspirin NSAID use conferred risk reductions for dysplastic Barrett's oesophagus, regardless of the control series. We excluded effect modification by known risk factors as an explanation for these null findings. CONCLUSIONS: We found little support for an inverse association between use of NSAIDs and Barrett's oesophagus. The question of whether or not these medications prevent the onset of Barrett's oesophagus remains open.

Early morbidity encountered in the dietary-related mouse model of Barrett's esophagus: a question of zinc?

Recently, a mouse model for Barrett's esophagus based on a zinc-deficient diet supplemented with deoxycholic bile acids has been published. The aim of this study was to attempt to reproduce these data and extend them by employing genetically modified mice and intraperitoneal iron supplementation. The study design encompassed six experimental groups (wild type, Apc-mutant and Smad4-mutant mice, with or without iron injections), with all animals fed with the zinc-deficient diet supplemented with deoxycholic bile acids. All treatments were started at 3-5 weeks of age (the majority [78%] at 5 weeks). Animals were scheduled for euthanasia at two distinct time points, namely at 3 and 6 months of age. All mice showed signs of considerable distress already 4 weeks after the start of the modified diets, and had to be euthanized before the first evaluation time point (mean age 9.3 weeks, range 5-15 weeks). No differences were observed between wild type and genetically modified mice, or between animals with or without iron supplementation. On histological examination, we could not detect any lesions (Barrett's esophagus-like or tumors) other than esophagitis. In the currently presented experimental settings, we were not able to reproduce the mouse model according to which Barrett's-like lesions could be detected in animals fed with the zinc-deficient diet supplemented with deoxycholic bile acids.

Risk of Barrett's oesophagus, oesophageal adenocarcinoma and reflux oesophagitis and the use of nitrates and asthma medications.

OBJECTIVE: To investigate the relationship between use of asthma medication and nitrates and risk of reflux oesophagitis, Barrett's oesophagus and oesophageal adenocarcinoma. MATERIAL AND METHODS: Data were collected on use of asthma medication and nitrates at least 1 year before interview from patients with reflux oesophagitis, Barrett's oesophagus and oesophageal adenocarcinoma. Associations between use of asthma medications and nitrates and the risk of reflux oesophagitis, Barrett's oesophagus and oesophageal adenocarcinoma were estimated using multiple logistic regression. RESULTS: Nine hundred and forty-one subjects were recruited: 230 reflux oesophagitis, 224 Barrett's oesophagus, 227 oesophageal adenocarcinoma patients and 260 population controls. Barrett's oesophagus patients were more likely than controls to have had a diagnosis of asthma (odds ratio 2.15, 95% confidence interval 1.15-4.03) and to have used asthma medications (odds ratio 2.13, 95% confidence interval 1.09-4.16). No significant associations were observed between use of asthma medication or nitrates and reflux oesophagitis or oesophageal adenocarcinoma. CONCLUSIONS: Gastro-oesophageal reflux symptoms appear to confound the association between asthma medication use and Barrett's oesophagus. However, it is possible that asthma medications may increase the risk of Barrett's oesophagus by other mechanisms.

Exogenous luminal nitric oxide exposure accelerates columnar transformation of rat esophagus.

Exposure of the esophageal mucosa to refluxed gastroduodenal contents is recognized to be an important risk factor for Barrett's esophagus (BE). At the human gastroesophageal junction, nitric oxide is generated luminally through the enterosalivary recirculation of dietary nitrate, and in cases with gastroesophageal reflux, the site of luminal nitric oxide generation could shift to the distal esophagus. The aim of this study is to investigate whether exogenous luminal nitric oxide could promote the development of BE in rats. Sodium nitrite plus ascorbic acid were administered to a rat surgical model of BE, in which the gastroduodenal contents were refluxed into the esophagus to generate exogenous luminal nitric oxide in the esophagus by the acid-catalyzed chemical reaction between the 2 reagents. The emergence of BE was evaluated histologically in the early phase (several weeks) after the surgery with or without exogenous nitric oxide administration. To elucidate the histogenesis of BE, CDX2, MUC2 and MUC6 expressions were investigated immunohistochemically. Coadministration of sodium nitrite plus ascorbic acid significantly accelerated the timing of emergence and increased the area of BE compared with controls. Administration of either reagent alone did not show any promotive effects on BE formation. Immunohistochemically, the columnar epithelium thus induced was similar to the specialized intestinal metaplasia in human BE. The results of this animal model study suggest that exogenous luminal nitric oxide could be involved in the pathogenesis of the columnar transformation of the esophagus. Further studies in human are warranted.

Role of nitrites in the genesis of adenocarcinoma associated with Barrett's esophagus.

BACKGROUND: Barrett's esophagus (BE) is one of the complications of gastroesophageal reflux disease (GERD) and a premalignant condition. It consists of a process of replacement of the squamous epithelium of the esophagus by intestinal columnar epithelium containing goblet cells, known as specialized intestinal metaplasia with goblet cells, and several factors have been related to its pathogenesis. The objective of this study was to evaluate an experimental model of duodenogastroesophageal reflux and the effect of ingestion of sodium nitrite solution on the genesis of adenocarcinoma associated with Barrett's esophagus. MATERIALS AND METHODS: Sixty male Wistar rats were divided into four groups. Twenty were not submitted to surgery and served as controls (10 animals ingesting only water and 10 ingesting water plus a solution of sodium nitrite), while the remaining 40 animals were submitted to side-to-side duodenogastroesophageal anastomosis (20 animals ingesting only water and 20 ingesting water plus the sodium nitrite solution). The Vienna classification for dysplasia and adenocarcinoma was used in the analysis of results. RESULTS: After 42 weeks of observation, Barrett's esophagus was found in 26.3% (5/19) of the animals submitted to surgery that had not ingested nitrites compared to 72.3% (13/18) of the animals in the group submitted to surgery and given nitrites. Six cases of adenocarcinoma (33.3%) were also found in this latter group. Barrett's esophagus was not found in any of the animals that were not submitted to surgery. Categories 2, 3 and 5 of the Vienna classification were only found in the animals submitted to surgery that also received sodium nitrite (66.7%). CONCLUSION: The ingestion of sodium nitrite associated with duodenogastroesophageal reflux plays an important role in the genesis of adenocarcinoma associated with Barrett's esophagus.

Bile acids and Barrett's oesophagus: a sine qua non or coincidence?

BACKGROUND: Barrett's oesophagus (BO), a premalignant condition associated with the development of oesophageal adenocarcinoma (OAC), is thought to be a consequence of chronic duodeno-gastro-oesophageal reflux. Of the refluxates, bile acids, either alone or in combination with acid, are probably the most important. METHODS: Analysis of the literature on the role played by bile acids in inducing BO and/or progression to OAC. RESULTS: Combined pH and Bilitec 2000 (as a measure of bile reflux) monitoring and oesophageal aspiration studies in humans suggest a combined role for bile acids, particularly taurine conjugated bile acids, in causing oesophageal mucosal injury. Evidence from animal models has demonstrated that duodenal juice alone is also able to induce BO and/or OAC. Likewise, ex vivo studies with biopsies from BO patients show that increased proliferation and cyclo-oxygenase-2 expression are present after a pulsed exposure to acid or conjugated bile acids, but not if acid and bile acids are combined. Proton-pump inhibitors (PPIs) have been shown to decrease the biliary component of the refluxate. There is some evidence that PPIs are able to reduce neoplastic progression in BO. On the other hand, chronic PPIs can also stimulate bacterial overgrowth, which can result in increased production of secondary bile acids, particularly deoxycholic acid, in the stomach. Deoxycholic acid has been demonstrated to have a tumour-promoting capacity. CONCLUSIONS: It is unknown what factors of the refluxate (acid and/or bile) induce BO and/or promote carcinogenesis, but there is evidence that secondary bile acids play a role. A better understanding of the molecular steps involved in the induction of BO, and the role of bile acids herein, may identify targets at which preventive therapies can be directed.

Chronic acid exposure leads to activation of the cdx2 intestinal homeobox gene in a long-term culture of mouse esophageal keratinocytes.

To explore mechanisms whereby Malpighian keratinocytes can transdifferentiate into an intestinal-like epithelium, as observed in the early steps of Barrett's esophagus (BE) development, long-standing cultures of esophageal keratinocytes derived from normal mouse esophageal explants were developed. These cells were able to form multilayers and to differentiate on filter support by the formation of differentiated layers of basal cells (cytokeratine 14 positive) on which secondary suprabasal cell layers (cytokeratine 4 positive) spontaneously developed. Thus, these cultured cells, referred to as P3E6, reproduced, at least in part, the proliferation and stratification pattern existing in the normal esophagus. Because chronic exposure to acid pH is known to be a critical factor for BE development, culture medium at pH 3.5 was added into the apical chamber of cell cultures. This led to a decrease in the overall number of cells but it did not affect cell proliferation. Furthermore, external acid environment triggered expression of the GFP reporter gene fused downstream of the cdx2 intestinal homeogene regulatory sequences in P3E6 transfected cells. Expression of the endogenous CDX2 protein, detected by western blot and immunocytochemical analysis, correlated with promoter activation. These findings demonstrate that chronic exposure of esophageal keratinocytes to acid pH induces transcription of cdx2, an intestinal specific homeobox gene known to play a critical role in the differentiation and maintenance of intestinal epithelial functions. The results suggest that chronic acid exposure can modify the fate of P3E6 esophageal keratinocytes towards an intestinal program. This can be a key step in the development of intestinal metaplasia often observed in esophagus-cardia junction.

Barrett's esophagus and chemotherapy, a case report.

There have been few reports of Barrett's esophagus associated with chemotherapy in children. We report the case of a 3-year-old patient diagnosed with acute lymphoblastic leukemia who developed Barrett's esophagus after BMF-90 chemotherapeutic regimen. A stricture appeared as a complication of Barrett's metaplasia and Nissen fundoplication was performed. Symptoms improved shortly after surgery and regression of Barrett's esophagus was observed 2 years later. Children treated with antileukemic chemotherapy may develop Barrett's esophagus without previous clinical apparent gastroesophageal reflux. Endoscopic surveillance has been advised in these patients. Barrett's esophagus may regress after antireflux surgery.

Metabolism of carcinogenic nitrosamines in the rat and human esophagus and induction of esophageal adenocarcinoma in rats.

The mechanism is discussed by which certain nitrosamines induce esophageal papillomas and squamous cancer in rats, and some evidence is presented for the view that nitrosamines also induce the same cancer in humans, especially in China and South Africa. Studies on the metabolism of nitrosamines by cytochrome P450 isozymes in rat and human esophagus, including the activation reactions of formaldehyde and pentaldehyde formation from methyl-n-amylnitrosamine (MNAN), are reviewed. These reactions are catalyzed by microsomes from the rat and human esophagus, probably because these microsomes contain specific cytochrome P450 isozymes. Evidence is reviewed for the occurrence of nitrosamines related to MNAN in fungus-infected corn. The incidence of esophageal adenocarcinoma is rising in Western countries. The precursor lesion, Barrett's esophagus, is associated with colon cancer, suggesting a role for bile salts in the induction of the esophageal tumor. Studies are described in which rats were subjected to esophago-duodenostomy (joining the duodenum to the esophagus) and then treated with nitrosamines that normally induce esophageal squamous cancer. Adenocarcinomas of the lower esophagus were induced as well as Barrett's esophagus (under one set of conditions). Feeding a high-fat diet with this system increased the incidence of esophageal adenocarcinoma. This tumor was not induced when the operation was changed to esophago-gastroplasty (widening the lower esophageal sphincter). These results support a role of reflux of duodenal contents (including bile and pancreatic juice) rather than of gastric contents in the etiology of human esophageal adenocarcinoma.

Is chemotherapy associated with development of Barrett's esophagus?

Columnar-lined or Barrett's esophagus is a premalignant condition. It is almost unvariably due to chronic gastroesophageal reflux. Since there are some reports that Barrett's esophagus can be induced by chemotherapy, we investigated 20 male patients, treated with chemotherapy for testicular cancer, and 18 female patients, treated with high-dose chemotherapy for breast cancer. Only one patient in the testicular cancer group had Barrett's esophagus of the circumferential type, in addition to typical reflux esophagitis and a hiatal hernia four years after chemotherapy. In the breast cancer group one patient had an indeterminate junction. Our results do not support the hypothesis that chemotherapy poses a substantially increased risk for the development of Barrett's esophagus.

Barrett's esophagus: lack of association with adjuvant chemotherapy for localized breast carcinoma.

This study was designed to prospectively, endoscopically, and histologically determine the prevalence of Barrett's esophagus in patients who had received adjuvant chemotherapy for localized breast carcinoma. Fifteen white women who received cyclophosphamide, methotrexate, and 5-fluorouracil (N = 8) or cyclophosphamide, adriamycin, and 5-fluorouracil (N = 7) chemotherapy underwent esophagogastroduodenoscopy and esophageal biopsy a mean 31.1 months (median, 11 months) after completion of full-course chemotherapy. Twelve of 15 patients had experienced oral ulcerations, diarrhea, or odynophagia during chemotherapy. In no patient was Barrett's esophagus identified endoscopically or histologically. This study fails to demonstrate an increased prevalence of Barrett's esophagus in breast carcinoma patients who had received adjuvant chemotherapy.