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10.1245/s10434-007-9387-7
Annals of Surgical Oncology 14:2045-2055 (2007)
© 2007 Society of Surgical Oncology
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Original Article

Alterations in Manganese Superoxide Dismutase Expression in the Progression From Reflux Esophagitis to Esophageal Adenocarcinoma

Yan Li, PhD1, John M. Wo, MD2, Ruifeng R. Su, BS1, Mukunda B. Ray, MD, PhD3 and Robert C. G. Martin, MD1

1 Division of Surgical Oncology, University of Louisville School of Medicine, 315 E Broadway #312, Louisville, Kentucky 40202
2 Division of Gastroenterology/Hepatology, University of Louisville School of Medicine, 315 E Broadway #312, Louisville, Kentucky 40202
3 Department of Anatomical Pathology, University of Louisville School of Medicine, 315 E Broadway #312, Louisville, Kentucky 40202

Correspondence: Address correspondence and reprint requests to: Robert C. G. Martin, MD; E-mail: robert.martin{at}louisville.edu


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Background: Comprehensive understanding of the basic mechanisms in the progression of esophagitis, Barrett esophagus (BE), and esophageal adenocarcinoma (EAC) is urgently needed to develop a management strategy for an effective screening of BE and management of EAC. The aim of this study is to provide a detailed insight of the histology and the cellular and molecular events associated with the genesis of BE and EAC under the esophagoduodenal reflux conditions.

Methods: Esophagoduodenal anastomosis (EDA) was performed on rats. Animals were weighed weekly and killed after 1, 2, 3, 4, 5, and 6 months. The entire esophagi were examined for macroscopic and microscopic changes and for manganese superoxide dismutase (MnSOD) expression, and TUNEL (terminal deoxynucleotidyl transferase dUTP nick-end labeling) assay was performed.

Results: Morphological transformation from esophagitis (100% of animals) to BE (66% of animals) to EAC was observed after 3 months. There was marked loss of MnSOD expression in animals with esophagitis and BE at 1 and 2 months, with an increase in expression during the transformation to dysplasia and EAC. Increased proliferation and apoptosis was observed and reached a peak at months 1 and 2. Greatly increased levels of 8-hydroxy-deoxyguanosine was found during the progression to EAC.

Conclusions: The morphological transformation of the esophageal mucosa is an adaptive process, and it is an important foundation for the transdifferentiation of BE and cancer. The significant loss of MnSOD expression to achieve BE and then the adaptive increase in expression to achieve dysplasia and EAC during this transformation may represent a predictive marker in identifying patients who will progress from BE to EAC.

Key Words: Superoxide dismutase • Barrett esophagus • Esophageal adenocarcinoma


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Gastroesophageal reflux disease (GERD) is one of the most common diseases; it affects more than 20% of the adult American population.1,2 The repetitive reflux causes progressive mucosal damage, leading to reflux esophagitis. Approximately 10% to 20% patients with chronic reflux develop Barrett esophagus (BE), defined by the replacement of the normal esophageal squamous lining by a specialized intestinal metaplasia.3,4 Patients with BE are at 30 to 40 times higher risk in developing esophageal adenocarcinoma (EAC) compared with the general population.3,5 EAC is one of the most rapidly rising cancers in white men in the United States,6,7 with a 5-year survival of only 15% after diagnosis.8 Nearly all EAC has been demonstrated to arise from BE, a premalignant disease caused by GERD.

It remains unclear why some BE patients develop EAC and some do not. Endoscopic surveillance remains the only current clinical strategy in evaluating the risk of BE.9 However, the inability to identify a definitive high-risk population whose disease will progress has limited the efficacy and cost-effectiveness of endoscopic screening. A comprehensive understanding of the basic mechanisms in the progression of esophagitis, BE, and EAC is urgently needed to develop a management strategy for BE and EAC. It is now widely accepted that EAC progresses in a stepwise fashion from esophagitis to metaplasia to dysplasia to EAC.10 The histologic changes leading to BE and EAC are accompanied by alterations at the cellular and molecular levels; however, specific markers for these alterations have not been universally defined.

Abnormalities in apoptosis, proliferation, and differentiation are common cellular events in BE and EAC.11–13 Studies have demonstrated an increase in proliferation in dysplastic and carcinomatous tissue compared with metaplastic tissue in BE and EAC patients.14,15 In contrast to the proliferation, there has been a decrease in apoptosis during the progression from BE to EAC. It is speculated that through the increased proliferation rate of premalignant and cancerous cell types, a clonal expansion of metaplastic or neoplastic cells occurs, leading to the growth of BE or EAC, and the abnormal cellular events is mainly in response to GERD. Recent studies have shown that GERD-induced chronic oxidative stress could play an important role in the progression of BE and EAC.16,17 Oxidative damage has been proposed to be closely related to reflux esophagitis and BE, with an increase in reactive oxygen species and lipid peroxidation corresponding to the severity of esophagitis and BE.18 Several studies also reported that antioxidants such as manganese superoxide dismutase (MnSOD) are depressed during the transition from esophagitis to BE.19,20 Reactive oxygen species causes DNA injury such as strand breakage, alterations in guanine and thymine bases, and DNA cross-linkage.21,22 Oxidative DNA damage may contribute to the accumulation of genetic damage, and the accumulation of genetic and epigenetic aberrations produces one or more clones with metaplastic and/or malignant potential.23

The determination of these cellular events and molecular events that occur in the transition from normal esophageal squamous mucosa to metaplasia to dysplasia to EAC will lead to better understanding of the process of the transformation to EAC. This knowledge will lead to a better biomarker that may diagnose and assess cancer risk. The esophagoduodenal anastomosis (EDA) in rat is the most commonly used surgical model to produce duodenogastroesophageal reflux. This model produces reflux esophagitis to BE to BE with dysplasia to EAC.24–26

In this study, we proposed to perform a time-course observation regarding the alteration in cellular proliferation, apoptosis, and oxidative stress in esophageal epithelium of EDA rat during the transformation of columnar lined epithelium (CLE) and EAC. The aim of this study is to provide a detailed insight of the cellular and molecular events associated with the genesis of BE and EAC under the esophagoduodenal reflux condition.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Animals and Treatment
Eight-week old Sprague-Dawley rats (Harlan, Indianapolis, IN) were housed three per cage, given commercial rat chow and tap water, and maintained on a 12-hour light-dark cycle. They were allowed to acclimate for 2 weeks before surgery. Solid food was withdrawn 1 day before and 1 day after surgery. EDA was performed on rats according to the operating procedure described previously.27 In brief, the animals were anesthetized with 60 mg/kg sodium pentobarbital, and the pedal reflex was used to monitor the depth of anesthesia. The gastroesophageal junction was ligated flush with stomach and the distal esophagus transected proximal to the ligature. An enterotomy (4 mm) was made 1 cm distal to the pylorus on the antimesenteric border. The distal esophagus was then anastomosed to the duodenal enterotomy with accurate mucosal to mucosal opposition.

Postoperatively, the animals were provided water after 2 hours and rat chow the next day. The animals were weighed weekly. Rats were killed after EDA for 1, 2, 3, 4, 5, and 6 months, then compared with sham nonoperated controls, which we have demonstrated in previous reports to be equivalent to sham-operated control groups.28 The esophagi entire were collected and examined for macroscopic and microscopic changes, immunohistochemical staining, and TUN-EL (terminal deoxynucleotidyl transferase dUTP nick-end labeling) assay. The esophageal mucosal layer was stripped from the muscle layer, and samples of both layers were used to perform Western blot and enzyme activity measurements.

This study was approved by the Institutional Animal Care and Use Committee at the University of Louisville.

Histopathology
The entire esophagus was removed and opened longitudinally to look for evidence of gross abnormalities. All samples analyzed for this study were of esophageal tissue (.5 cm in length) from the distal part of the esophagus. Samples were fixed in 10% buffered formalin for 24 hours and transferred to 80% ethanol. The formalin-fixed esophagus was embedded in paraffin. Serial sections of 5 (µ m were mounted onto glass slides for histopathological and immunohistochemical analysis. Hematoxylin and eosin–stained slides were obtained for each rat. Evidence for reflux esophagitis was identified in the esophageal epithelium, such as the infiltration of inflammatory cells, basal cell hyperproliferation, papillae hypertrophy, dilation of venules, ingrowth of the capillaries, epithelial sloughing, and ulceration.29

TUNEL Assay
ApopTag in situ apoptosis detection kit (Intergen Company, Purchase, NY) was used to detect the apoptotic cells according to a procedure reported previously.30 Five-micron-thick sections were cut from the paraffin blocks. After paraffin was removed from the samples, they were rehydrated, and endogenous peroxidase was blocked with H2O2 in methanol for 20 minutes. The sections underwent proteinase K digestion for 15 minutes. DNA fragments were tailed with digoxigenin-dUTP along with antidigoxigenin antibody conjugation with horseradish peroxidase along with the substrate (DAB-H2O2) to develop a brown color. The TUNEL-positive epithelial cells were counted against negative cells under a light microscope at a magnification of x 40, and six visual epithelium fields were chosen on each slide. All sections from each animal were examined. An apoptotic index (the number of epithelial nuclei labeled by the TUNEL method divided by the number of total epithelial nuclei) was calculated. A systematic approach was taken for analysis in which randomly generated visual fields were chosen on each slide to avoid investigator bias.

Immunohistochemical Assay
Immunohistochemical assays were performed to detect proliferating cell nuclear antigen (PCNA), 8-hydroxy-deoxyguanosine (8-OH-dG), and MnSOD. Immunohistochemical staining was carried out on the paraffin-embedded material with the Dako EnVi-sion+System Kit (Dako Corporation, Carpinteria, CA). In brief, paraffin was removed from the sections and the sections were hydrated. The slides were washed with a Tris buffer, and peroxidase blocking was performed for 5 minutes.

After rewashing, the primary antibodies were applied for 30 minutes. The antibodies included multiclonal Ab-PCNA, multiclonal Ab-8-OH-dG, and multiclonal Ab-MnSOD (Santa Cruz Biotechnology, Santa Cruz, CA). After rewashing, 30-minute incubation was performed in the Dako Envision–labeled polymer, and the substrate-chromogen solution (di-aminobenzidine) was added as a visualization reagent. Finally, the slides were counterstained with methyl green. A negative control was included in each run. The PCNA-positive epithelial cells were counted against negative cells under a light microscope at a magnification of x 40. Just as for the apoptotic index, six visual fields were chosen on each slide, and the proliferation index was calculated as a ratio of the number of PCNA positive epithelial nuclei to the number of total epithelial nuclei. 8-OH-dG staining and MnSOD were scored under microscopy. Scoring immunohistochemical staining was performed as follows: 0, negative staining; 1, weak staining; 2, moderate staining; 3, strong staining. Staining was evaluated in both the mucosa and the muscularis of the esophagus with the ability to give a maximum score of 6.

Western Blot Analysis of MnSOD Expression
Western blot was performed to determine the MnSOD protein expression in the esophageal mucosal layer and muscle layer. In brief, total protein is isolated from fresh tissue samples by homogenization in ice-cold buffer containing 20 mM of HEPES (pH 7.5), 1.5 mM of MgCl2, 20 mM of dithiothreitol, .4 M of NaCl, 20% glycerol, .5 mM of phenylmeth-ylsulfonyl fluoride, and .5 mM of leupeptin at 4° C. Insoluble cellular material was removed by micro-centrifugation at 16,000 x g for 5 minutes, and total protein was determined spectrophotometrically. The protein samples were separated by sodium dodecyl sulfate–polyacrylamide gel electrophoresis and subsequently transferred to the nitrocellulose membrane for Western blot test, as described previously.31

Statistical Analysis
Student’s t-tests assuming unequal variance were performed. The results are expressed as mean values ± standard deviation. Comparisons were made among the bile perfusion groups and saline control groups by analysis of variance. A P value of less than .05 was considered statistically significant.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Fifty rats underwent EDA, and four early deaths (8%) occurred within 48 hours after surgery, one due to anesthesia, two to esophageal suture blockage, and one to an unknown cause. Three later deaths (6%) occurred at 1 month, 5 months, and 5.5 months for the reasons of esophageal dilation in one and trans-mural inflammation with esophageal necrosis in two. The remaining rats survived and made up the study groups.

Esophageal Pathogenesis
In the animals with EDA for 1 and 2 months, 50% of the distal esophagus was dilated, mucosal layer was thickened and friable, and the mucosal surface was grossly irregular compared with the nonoperated controls consistent with esophagitis. In the animals with EDA for 3, 4, 5, and 6 months, the gross appearance was similar as the 1- and 2-month rats, but was more extensive, with three-fourths of distal esophagus involved. In addition, 8 of 15 animals with diagnoses of EAC had visible tumors, five rats (one from month 3, two from month 4, and two from month 6) had single large tumors. The tumors measured .2 to .5 cm in diameter, with ulceration and necrotic foci. Three rats (one from month 4, one from month 5, and one from month 6) demonstrated multiple tumor foci.

The time course of morphological changes in esophageal epithelium of rats with EDA is illustrated in Fig. 1Go. Squamous hyperplasia with papillomatosis was seen in all rats with EDA, and the degree of extension of the lamina propria papillae within the esophageal mucosa was increased along the time course of the EDA model. The reflux esophagitis were quantified by the Hetzel grading system32 (Table 1Go). The grading score in each EDA group slightly increased from month 1 through month 6, but the differences were not statistically significant among the EDA rats. The incidences of intestinal metaplastic cell–lined esophageal mucosa increased from month 3 to month 6, with all CLE occurring at the distal esophagus, extending upward from the EDA. The representative histology of BE and EAC by hema-toxylin and eosin staining is illustrated in Fig. 2Go, with goblet cell identification in the specimens of BE and EAC. Alcian blue and periodic acid–Schiff staining were also used to detect acid and neutrophil mucins secretion. Alcian blue highlighted the goblet cells in BE and EAC, while periodic acid–Schiff staining highlighted the interstitial portion (Fig. 3Go).


Figure 1
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  		FIG. 1. Histological changes of esophageal epithelium after esophagoduodenal anastomosis (EDA). N, normal esophagus; W, week; M, month; BE, Barrett esophagus; EAC, esophageal adenocarcinoma. Papillae elongation and mucosa ulceration were seen in the esophageal epithelium after EDA, while papillomatosis with multilayer squamous cells predominated after EDA 3 months (hematoxylin and eosin; original magnification, x 200).

 

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  		TABLE 1. Histology of rat esophagi after esophagoduodenal anastomosis (EDA)a
 

Figure 2
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  		FIG. 2. Appearances of BE and EAC in rat esophageal epithelium after esophagoduode-nal anastomosis (EDA). BE, Barrett esophagus; EAC, esophageal adenocarcinoma. Specific intestinal metaplasia with identifica-tion of goblet cells showed in the distal esop-hageal epithelium; some squamous epithelium also can be seen among the columnar cells. Esophageal adenocarcinoma was detected with the identification of goblet cells in the submucosa. Pieces of squamous epithelium can be seen among the carcinoma.

 

Figure 3
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  		FIG. 3. Identification of goblet cell in Barrett esophagus (BE) and esophageal adenocarci-noma (EAC) with Alcian blue staining. Arrow, goblet cell; arrowhead, Alcian blue–positively stained cell.

 
Proliferation and Apoptosis of Esophageal Epithelial Cells
Esophageal epithelial cell apoptosis and proliferation were assessed under light microscopy. Compared with the nonoperated controls, EDA rats showed far greater TUNEL-positive staining in esophageal epithelium, and the positive nuclei were found mostly in the keratinized layer (Fig. 4Go). Minimal TUNEL-positive cells were found in the submucosa and muscle layers. The apoptotic index in esophageal epithelium with diagnoses of esophagitis (corresponding to EDA 1 week and EDA 2 weeks; Fig. 4Go) and BE (corresponding to EDA 1 month and EDA 2 months, Fig. 4Go) statistically significantly increased compared with the nonoperated controls (P < .05). The apop-totic index in esophageal epithelium with diagnosis of EAC (corresponding EDA 5 months and 6 months, Fig. 4Go) was decreased compared with that in esoph-agitis and BE (Fig. 4Go). PCNA staining was used as an index of the esophageal mucosa proliferation. A number of PCNA-positive cells were seen within the esophageal epithelium, especially in the basal layer in rats after EDA. The proliferating index was signifi-cantly increased in the esophageal mucosa with EDA compared with the nonoperated controls (P < .05); however, we did not observe a disease- or time-dependent increase of proliferation index (Fig. 5Go).


Figure 4
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  		FIG. 4. Apoptosis in rat esophageal epithelium after esophagoduodenal anastomosis (EDA). N, normal esophagus; W, week; M, month; BE, Barrett esophagus; EAC, esophageal adenocarcinoma. TUNEL staining (original magnification, x 200) and apoptotic index (data presented as mean ± SD, *P < .05 vs. normal control; # P < .05 vs. BE).

 

Figure 5
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  		FIG. 5. Proliferation in rat esophageal epithelium after esophagoduodenal anastomosis (EDA). N, normal esophagus; W, week; M, month; BE, Barrett esophagus; EAC, esophageal adenocarcinoma. Proliferating cell nuclear antigen staining (original magnification, x 200) and apoptotic index (data presented as mean ± SD, *P < .05 vs. normal control).

 
Oxidative Damage in the rat Esophagus
The study of two oxidative damage parameters was performed in the esophageal specimens after EDA, 8-OH-dG for oxidative damage to DNA and MnSOD for loss of antioxidative capacity. The levels of 8-OH-dG and MnSOD in the esophageal epithelium were evaluated by immunohistochemical staining score. As compared with the nonoperated controls, the level of 8-OH-dG was statistically significantly increased in the esophageal epithelium with the diagnoses of esopha-gitis, intestinal metaplasia, and EAC. The level of MnSOD was also increased in the malignant epithelium of EAC, but it was decreased in the esophagitis epithelium and intestinal metaplastic epithelium (Table 2Go). A time-course study of MnSOD expression was performed in the esophageal tissue from mucosal layer by Western blot analysis. There was a high loss of MnSOD expression in the rats after EDA 1 and 2 months, corresponding to the histologic features of esophagitis and BE. However, there were markedly increased expression of MnSOD in the rats after EDA 3, 4, 5, and 6 months compared with the nonoperated (Fig. 6Go) controls, corresponding to BE with dysplasia and EAC.


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  		TABLE 2. Expression levels of 8-OH-dG and MnSOD by immunohistochemistrya
 

Figure 6
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  		FIG. 6. MnSOD expression in rat esophageal epithelium after esophagoduodenal anastomosis (EDA). N, normal esophagus; M, month.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
This study demonstrated that in a rat model, duodenogastroesophageal reflux potentiates the development of reflux esophagitis, intestinal metaplasia, and EAC. The preferential location of intestinal metaplasia and carcinoma is in the lower esophagus, corresponding to location in humans and relates to the greater bile exposure of the distal esophagus. An increased rate of proliferation along with a decreased rate of apoptosis was observed in the epithelium of metaplasia and EAC compared with normal squamous epithelium. A decreased level of MnSOD and an increased level of 8-OH-dG in esophageal epithelial cells were associated with developments of intestinal metaplasia and cancer. MnSOD expression was decreased during the initial transdifferentiation phase to the CLE of BE, with a subsequent increase in expression during the progression to EAC (Fig. 6Go).

Recent studies indicate that MnSOD, one member of SOD family and an essential enzyme for life, plays an important role in the protection against radiation-induced esophagitis and animal mortality after radiation exposure.33,34 We have previously shown that expression changes occur in MnSOD from normal esophagus to BE to EAC. We found that MnSOD expression decreased in esophageal epithelial cells that had undergone changes from normal squamous esophageal cells to Barrett metaplasia.19 This information implies that the depletion of MnSOD in esophageal epithelium by gastroesophageal reflux could be an important pathogenesis in reflux esoph-agitis, BE, and EAC.

The data presented here demonstrate a transient loss of MnSOD expression during repetitive oxidative stress that may lead to increased oxidative damage and the loss of the protective response to form a columnar epithelium of BE. This response, however, leads to epigenetic aberrations with malignant potential that may be demonstrated with an adaptive increase in MnSOD, which may lead to the apoptotic stabilization and malignant transformation. Currently, the association of gastroesophageal reflux and the metaplasia to dysplasia to EAC sequence has been firmly established.35,36 However, the pathogenesis underlining intestinal metaplasia and esophageal carcinogenesis are poorly understood. Duodeno-esophagostomy, known as the EDA model, is the most commonly used surgical model to mimic the reflux situation by introducing mixed gastric and duodenal contents into the esophagus. A number of reports that used the EDA animal model confirm the result of the metaplasia to dysplasia to EAC sequence, but many of these studies used supplemental agents to promote the development of metaplasia or cancer in the EDA model that could affect the process of metaplasia to dysplasia to EAC sequence.37,38 Thus, a detailed analysis regarding the histological change and cellular events under EDA condition only is needed.

In this study, we reproduced the EDA model in rats without the use of any supplementation such as carcinogens, fat, or iron to observe the development of intestinal metaplasia and EAC under the gastro-duodenal refluxate condition. The rates of intestinal metaplasia and EAC were similar to those reported by others.39 In addition, the time-course study revealed a dynamic morphological change, which presented an adaptive process in esophageal mucosa after EDA. Gastroduodenal reflux damage to the esophageal mucosa causes esophagitis with the his-tological features of ulceration and papillomatosis of the squamous epithelium, which is demonstrated and predominates in the EDA rat at 3 months. However, in rats with EDA that develops after more than 3 months, the occurrence of intestinal metaplasia and esophageal carcinoma develops with the architectural feature presenting a dominant papillomatosis. This is quite different from that observed in EDA rat within 3 months. Replacement by columnar epithelium occurs in resistance to this repeated reflux injuries. Benign pedunculated squamous polyps are frequently found in the specimen along with the intestinal metaplasia and esophageal carcinoma, and they provide evidence that the morphological transformation of esophageal epithelial cell is associated with BE pathogenesis and esophageal carcinogenesis.

An important issue addressed in this study is the cellular events of proliferation and apoptosis in esophageal epithelium during the morphological transformation to multiple-layer squamous epithelia and the progression to intestinal metaplasia and esophageal carcinoma. Inhibition of apoptosis in BE and EAC has been reported in many studies from both animal and clinical studies,15,40,41 but our data for the first time demonstrate a time-course change of these two important cellular events during pathohis-tological severity and the metaplasia to dysplasia to EAC sequence. The proliferation index in the EDA rat reached a plateau at month 2 and demonstrated no statistically significant difference among benign pedunculated squamous polyps, intestinal metaplastic epithelium, and cancerous epithelium. In contrast to the proliferation index, the apoptosis index reached a peak at 1 month and decreased during the subsequent time points. Inhibition of apoptosis was found after 2 months in all esophageal epithelium with papillomatosis, intestinal metaplasia, and cancer. The increased indexes of proliferation and apoptosis in earlier EDA stage implied that the cells are undergoing an essential assessment mechanism to determine whether it is healthy enough to proceed to the next stage of cycle or whether it should permit cell cycle death.42 The inhibition of apoptosis in the later EDA time points indicate that only those cells that develop antiapoptotic mechanisms can have the opportunity to follow the metaplasia to dysplasia to EAC sequence.30

We have observed a dynamic change of histology and cellular events after EDA in rat esophagus. Our results suggest that the morphological transformation of esophageal mucosa is an adaptive process, and that it is also an important foundation for the transdifferentiation of intestinal metaplasia and cancer. The alternation of proliferation and apoptosis provides a balance mechanism of esophageal mucosa in response to the reflux injury after EDA, and inhibition of apoptosis is a high risk factor for intestinal metaplasia and cancer. Changes in MnSOD expression represents a potential marker that could be used to better identify the higher-risk patients with BE.

Received for publication September 12, 2006. Accepted for publication October 26, 2006.


   REFERENCES
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 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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