Oxidative damage in an esophageal adenocarcinoma model with rats
Xiaoxin Chen,
Yu Wei Ding,
Guang-yu Yang,
Flordeliza Bondoc,
Mao-Jun Lee and
Chung S. Yang1
Laboratory for Cancer Research, College of Pharmacy, Rutgers University, 164 Frelinghuysen Road, Piscataway, NJ 08854, USA
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Abstract
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Oxidative damage has long been related to carcinogenesis in human cancers and animal cancer models. Recently a rat esophageal adenocarcinoma (EAC) model was established in our laboratory by using esophagoduodenal anastomosis (EDA) plus iron supplementation. Our previous study suggested that iron supplementation enhanced inflammation and the production of reactive nitrogen species in the esophageal epithelium, which could contribute to esophageal adenocarcinogenesis. Here we further characterized oxidative damage in this model. We were particularly interested in how excess iron was deposited in the esophagus, and which cells were targeted by oxidative damage. Male SpragueDawley rats received iron supplementation (50 mg Fe/kg/month, i.p.) starting 4 weeks after EDA. The animals were killed at 11, 30 or 35 weeks after surgery. EAC appeared as early as week 11 after surgery, and increased over time, up to 60% at 35 weeks after surgery. All EACs were well-differentiated mucinous adenocarcinoma at the squamocolumnar junction. Iron deposition was found at the squamocolumnar junction and in the area with esophagitis. Esophageal iron overload could result from transient increase of blood iron after i.p. injection, and the overexpression of transferrin receptor in the premalignant columnar-lined esophagus (CLE) cells. Oxidative damage to DNA (8-hydroxy-2'-deoxyguanosine), protein (carbonyl content) and lipid (thiobarbituric acid reactive substance) in the esophagus was significantly higher than that of the non-operated control. CLE cells were believed to be the target cells of oxidative damage because they overexpressed heme oxygenase 1 and metallothionein, both known to be responsive to oxidative damage. We propose that oxidative damage plays an important role in the formation of EAC in the EDA model, and a similar situation may occur in humans with gastroesophageal reflux and iron over-nutrition.
Abbreviations: 8-OH-dG, 8-hydroxy-2'-deoxyguanosine; CLE, columnar-lined esophagus; EAC, esophageal adenocarcinoma; EDA, esophagoduodenal anastomosis; HO1, heme oxygenase 1; MT, metallothionein; ROS, reactive oxygen species; TBARS, thiobarbituric acid reactive substance; TfR, transferrin receptor.
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Introduction
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Esophageal adenocarcinoma (EAC) has received considerable attention in recent years because of its rapid increase in incidence. Between 1976 and 1990, the incidence rate of EAC in the USA tripled, with a yearly increase of ~10%, which was the fastest increase of all the cancers (1,2). It now accounts for >50% of all the esophageal cancers, and affects about 10 000 people per year (3). EAC has extremely bad prognosis with a 5 year survival rate of ~10% (4); therefore, it is of great importance to understand the pathogenesis and develop strategies for the prevention of this deadly disease.
It is now clear that most of the EACs develop from a premalignant disease, columnar-lined esophagus (CLE), also known as Barrett's esophagus, characterized by the replacement of squamous epithelium in the esophagus by columnar epithelium (3,5). Most CLE is preceded by reflux esophagitis, which is a commonly seen clinical identity in the western countries, with >30% of the general population experiencing its symptoms at least once every month (6). Approximately 10% of all the reflux esophagitis patients will eventually develop CLE (5,7). According to a large autopsy study, the incidence of CLE in the general US population was estimated to be one out of 80 (8). The risk of EAC for CLE patients has been estimated to be about one in 100 patient-years, 30125 times higher than the general population (5,9).
Oxidative damage has been proposed to be closely related to reflux esophagitis, and a possible cause for CLE (10). Wetscher et al. (11) found that reactive oxygen species (ROS), as measured by chemiluminescence and lipid peroxidation, increased with the grade of esophagitis and were the highest in CLE. Anti-reflux surgery prevented the development of oxidative damage in the esophagus. Consistent with this idea is that ß-carotene had been shown to prevent, even reverse, the progression of human CLE (12). Epidemiological studies also indicated an inverse association between the intake of ß-carotene and the risk of EAC (13). The strong association between EAC and smoking may partially attribute to the fact that cigarette smoking stimulates endogenous pulmonary and vascular production of ROS, and depletes endogenous antioxidant defense mechanisms (14).
Esophagoduodenal anastomosis (EDA), also known as esophagoduodenostomy with rats is the most commonly used surgical model to produce duodeno-gastro-esophageal reflux. This model produces CLE and low incidence of EAC, and carcinogenesis can be enhanced by treatment with nitrosamines (1517). However, all the EDA rats developed iron-deficiency anemia due to iron malabsorption. Supplementation with iron dextran by i.p. injection in the rats prevented anemia after surgery, but greatly enhanced the incidence of EAC (16). Our previous study also showed that iron-supplemented EDA rats had significantly higher levels of inflammation, cell proliferation, inducible nitric oxide synthase (iNOS) and nitrotyrosine immunostaining, iron deposition, as well as EAC tumors in the distal esophagi than those without iron supplementation (18). Since iron is known to promote oxidative stress, this appears to be an excellent model to study the role of oxidative damage in esophageal adenocarcinogenesis.
In this study, we further determined the oxidative damage parameters and carcinogenesis in our EDA model with i.p. iron supplementation. We were particularly interested in finding out the target cells of oxidative stress. Blood and tissue iron levels were determined to investigate how excess iron was deposited in the esophagus. The data presented here further supported our hypothesis that oxidative damage plays an important role in the formation of EAC.
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Materials and methods
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Animals and treatment
Six-week-old male SpragueDawley rats from Taconic Farms (Germantown, NY) were housed two per cage, given commercial rat chow and water ad libitum, and maintained on a 12 h light/dark cycle. They were allowed to acclimate for 2 weeks prior to surgery. Solid food was withdrawn 1 day before and for 1 day after surgery. EDA was performed according to the procedure described previously (16). This procedure was approved by the Animal Care and Facilities Committee, Rutgers University (protocol #94017). The animals were given iron dextran i.p. (50 mg Fe/kg/month), starting 4 weeks after surgery and continuing for the duration of the experiment. Another group of animals was included as non-operated controls (Table I
). The animals were weighed weekly. Blood samples (200 µl) were taken from the orbital venous sinus in 10 animals of each group under anesthesia with metophane periodically during the experiment to monitor the iron nutritional status.
All the rats were killed as described previously (16). Special care was taken to separate the esophagus from the duodenum based on the suture line. For the animals killed at weeks 11 and 30, the esophagi were cut longitudinally, with half quickly frozen on dry ice and then stored at 80°C for the analysis of oxidative damage parameters, and the other half fixed in 10% buffered formalin for 24 h and then transferred to 80% ethanol. For the animals killed at week 35, all the esophagi were fixed in formalin. The formalin-fixed esophagus was Swiss-rolled, processed and embedded in paraffin. Five-micron sections were mounted onto glass slides and used for pathological, histochemical and immunohistochemical analyses.
Iron nutritional status analysis
Fresh whole blood and serum were used for determination of hemoglobin, total serum iron and transferrin saturation with kits from Sigma Diagnostic (St Louis, MO). Instructions from the manufacturer were followed with slight modifications.
Histopathology
Histopathological analysis was carried out on the hematoxylin and eosin stained slides (the first, twentieth and fortieth slides). Reflux esophagitis was diagnosed when there was infiltration of numerous inflammatory cells, basal cell hyperproliferation, dilation of venules, in-growth of the capillaries, epithelial erosion and ulcers. Esophagitis was graded according to the Hetzel grading system (19). CLE was diagnosed when there was any length of intestinal columnar epithelium containing a villiform surface, mucous glands and intestinal-type goblet cells above the blue prolene suture. Dysplasia was characterized by the partial loss of cell polarity and maturation, nuclear atypia and an increase in mitotic figures of the CLE cells. EAC was diagnosed when dysplastic columnar epithelial cells invaded through the basement membrane (20).
Iron histochemistry and immunohistochemical staining
Ferric iron in the tissue sections was detected using the Prussian blue staining plus intensification with diaminobenzidine (DAB) (21). The avidin-biotin-peroxidase complex method (Elite ABC kit; Vector Laboratories, Burlingame, CA) was used for immunohistochemical staining for transferrin receptor (TfR), heme oxygenase 1 (HO1), and metallothionein (MT). A monoclonal mouse anti-rat TfR (10 µg/ml; Serotec, Raleigh, NC), a rabbit polyclonal anti-HO1 (1:100; Affinity Bioreagents, Golden, CO), and a mouse monoclonal anti-MT (2 µg/ml; Accurate, Westbury, NY) were used. Sections were pre-treated with target unmasking fluid (Pharmingen, San Diego, CA) or with trypsin. Negative controls were established by replacing the primary antibody with PBS and normal serum.
Determination of oxidative damage
Epithelium of the lower half of the frozen esophagus was stripped off and used for all the biochemical analyses. Lipid peroxidation was determined using an HPLC-based thiobarbituric acid reactive substance (HPLC-TBARS) method, using malonaldehyde bis(diethyl acetal) (MDA; Sigma) as the standard (22). Lipid peroxidation was expressed as nmol MDA/mg tissue protein.
For the analysis of 8-hydroxy-2'-deoxyguanosine (8-OH-dG), genomic DNA was extracted from esophageal epithelium with a Wako Extractor WB kit (Wako Chemical, Richmond, VA), hydrolyzed and analyzed by an HPLC system equipped with a reverse-phase column (PFP column, 60A, 5 µm; Princeton Chromatography, Princeton, NJ), an electrochemical detector (400 mA; Coulchem II; ESA Co., Chelmsford, MA) and a UV detector (280 nm; Waters 440; Waters-Millipore). The amount was expressed as the molar ratio of 8-OH-dG to 2'-dG (8-OH-dG per 105 2'-dG). Commercial calf thymus DNA (Sigma; three 8-OH-dG residues per 105 2'-dG residues) was included for quality control.
Carbonyl content was determined with a spectrophotometric assay (23). In this study, extreme care was taken to avoid release of nucleic acids into the homogenate during vigorous homogenization, and streptomycin sulfate (1%) was added to remove nucleic acids. The amount of carbonyl content was expressed as nmol/mg protein.
Statistical analysis
The result on pathogenesis was analyzed by the
2 test. Other data were analyzed by the Student's t-test using the computer software Statview 4.2.
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Results
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Fifty-five rats underwent EDA, five (9%) died: one due to anesthesia, three due to blockage (esophageal stricture), and one due to unknown reason. Animals were killed at 11, 30 and 35 weeks after EDA. The body weights of the EDA rats were significantly lower than the non-operated control rats throughout the experiment (P < 0.05) (Figure 1A
).
Iron nutritional status
In the first 4 weeks after the surgery, iron nutritional status parameters (hemoglobin, total serum iron and transferrin saturation) dropped markedly, then rose after iron supplementation and gradually reached plateau levels (Figure 1BD
). Injection of 50 mg Fe/kg/month i.p. resulted in adequate hemoglobin levels, although the total serum iron and transferrin saturation were still significantly lower than those of the non-operated controls (P < 0.05). We also examined total serum iron and transferrin saturation immediately after the injection of iron dextran. Within 1224 h after i.p. injection of iron, total serum iron and transferrin saturation values peaked at 900 µg/dl and 0.70, respectively, and then decreased to the levels prior to iron injection within 3 days (Figure 1E and F
).
Histopathogenesis
The Hetzel grading system was used to quantify the reflux esophagitis in all the EDA rats (Table I
). The Hetzel grade of Group II slightly increased from week 11 to week 30 and week 35, but the difference was not statistically significant. All the CLE occurred at the distal esophagus, extending upwards from the esophagoduodenal anastomosis. All the EAC were well-differentiated mucinous adenocarcinomas, arising in the squamocolumnar junction area. The incidence of EAC increased from week 11 to week 30 and week 35 (Table I
). Some EAC tumors had remarkable sizes, as big as 1.5 cm in diameter. Small ulcers and necrotic foci were found on some tumors.
Iron deposition in the esophagus
Positive iron staining, as detected by Prussian blue staining with DAB intensification, was seen in macrophages not only in the submucous layer, but also those in the conical papillae of lamina propria (Figure 2A
). Substantial amount of staining was seen in the distal esophagus, especially the squamocolumnar junction, where inflammation was most severe and all the EAC arose. Some of the iron-positive macrophages were immediately adjacent to the CLE cells (Figure 2B
). Negative iron staining was observed in the esophagi of the non-operated controls. Interestingly, iron-positive cells were scarcely seen in the EAC, but mostly found along the edges of the tumors.


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Fig. 2. Histochemical staining of the `free' iron in rat esophagi after EDA and i.p. iron supplementation. Positive iron staining (dark) was seen in lamina propria (A) and at the squamocolumnar junction and along the edge of EAC tumor (B). Both images were taken from one case of group II at 11 weeks after surgery. Tu, EAC tumor; se, squamous epithelium of the esophagus; ce, columnar epithelium. Magnification x100.
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In addition to `free' iron, we further localized TfR that binds transferrin, the carrier for iron transportation. It is known that proliferating cells assimilate iron by overexpressing TfR (24). Many CLE cells in the squamocolumnar junction were found to over-express TfR, suggesting that these cells could assimilate a lot of iron in transferrin-bound form. Less positive staining was seen in columnar cells away from the squamocolumnar junction (Figure 3A and B
). Some EAC cells, macrophages and some columnar cells in the duodenal epithelial crypt and the cryptvillus junction were also found to express TfR. No TfR was observed in the superficial villus enterocytes of the duodenum, as reported previously (24). It was particularly interesting that most iron, either in the `free' or `bound' form, was localized in the squamocolumnar junction area, where all the EAC resided.


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Fig. 3. Overexpression of TfR in CLE after EDA and i.p. iron supplementation [(A) x40 and (B) x200]. Arrows show the positive staining (dark). Su, surgical suture; se, squamous epithelium of the esophagus.
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Oxidative damage in the rat esophagus
We measured three oxidative damage parameters in the lower part of the esophageal epithelia of EDA rats: TBARS for oxidative damage to lipid, 8-OH-dG for oxidative damage to DNA, and carbonyl contents for oxidative damage to protein (Table II
). As compared with the non-operated controls, the EDA rats had significantly higher levels of TBARS, 8-OH-dG and carbonyl content at 11 and 30 weeks after surgery. However, we did not observe time-dependent increase of these parameters in the EDA group.
To localize the cells subject to oxidative damages, we examined the expression of two known oxidative stress-responsive genes, HO1 and MT, by immunohistochemistry (25). Both CLE cells at the squamocolumnar junction, and EAC overexpressed both HO1 and MT (Figure 4AD
). Overexpression of MT in the basal cells of the esophagitic squamous epithelia was also observed.
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Discussion
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EDA in rats, which mimics the human situation by introducing mixed reflux of gastric and duodenal contents into the esophagus, is the most commonly used animal model to produce CLE and EAC. Iron malnutrition is a serious problem for EDA rats. Previous experience showed that many EDA rats could not survive a 30 week experiment without iron supplementation due to severe iron-deficiency anemia (16). After EDA, insufficient gastric acid resulted in the formation of insoluble ferric salt that is non-absorbable. Occult bleeding due to reflux esophagitis, and rapid passage of food through the duodenum where most iron was absorbed, may also contribute to the iron deficiency (26). Injection of 50 mg Fe/kg/month resulted in adequate hemoglobin and better health.
After i.p. injection of iron dextran, high short-term spikes of serum iron and transferrin saturation appeared at 1224 h. When the esophageal epithelium was hyperemic and edematous under gastroesophageal reflux, the iron dextran may easily exude out of the dilated capillaries into the interstitial tissue. In the meantime, macrophages in reticuloendothelial system can incorporate some `free' serum iron into siderophore, and carry them to the site of inflammation, where they are poorly re-used and deposited (27). This may explain why the EDA rats all had excess `free' iron deposited in the esophagus, especially in the squamocolumnar junction area where the esophagitis was most severe, while their total serum iron and transferrin saturation values were yet lower than those of the non-operated control rats.
On the other hand, the metabolism of the bound form of iron is well regulated by the expression of TfR and ferritin. After iron-binding transferrin gets into cells expressing TfR, the iron is normally stored in ferritin (28). Although incorporation of iron into transferrin and ferritin confers some immediate protection against iron-related toxicity, the iron in ferritin and transferrin are also readily released when stimulated (28,29). As a consequence, iron-related toxicity may still happen in cells where the iron was delivered and stored, and may even affect the adjacent cells (29). In the present study, many CLE cells, EAC cells and macrophages over-expressed TfR, suggesting that these cells assimilated a considerable amount of iron in transferrin-bound form and then stored it in ferritin (Figure 3A and B
). The premalignant CLE cells appeared not only surrounded by excessive amount of exogenous iron, but also overloaded with endogenous iron.
It is likely that local iron overload is directly responsible for the enhancement of carcinogenesis. In the present study, the incidence of EAC in the EDA groups on 50 mg Fe/kg/month was as high as 60% at 30 weeks after surgery. EDA itself only induced 7% of EAC in rats at 30 weeks after surgery in one study (15), and no EAC in another two studies (30,31). It is interesting that all the EACs were seen at the squamocolumnar junction of the esophagus. A few factors may contribute to this phenomenon: (i) columnar cells at the squamocolumnar junction tend to be highly proliferating to replace the damaged squamous epithelium. The turnover rate of the columnar cells is approximately five times that of squamous cells, and the columnar epithelium is more resistant to damage from acid and bile acid than squamous epithelium (32). (ii) The squamocolumnar junction has long been predicted to be a site of epithelial stem cells, since graft-versus-host disease after bone marrow transplantation had predilection for the squamocolumnar junction in gut (33). (iii) Substantial amounts of iron were deposited in this region, and both iron and chronic inflammation are known to induce oxidative damage. Extensive oxidative damage on the proliferating stem cells could increase the chance of developing EAC. Our previous study showed overexpression of iNOS and nitrotyrosine in the rat esophagus after EDA and iron supplementation (18). In the present study, 8-OH-dG, lipid peroxidation and carbonyl content in the esophageal epithelia were significantly higher in the EDA rats than the non-operated controls. A strong correlation between oxidative damage and esophageal adenocarcinogensis was suggested. Although we did not observe time-dependent increase of oxidative damage in the esophagus, it was quite possible that, at 11 and 30 weeks after surgery, oxidative damage had reached a plateau. In another oxidative damage-related carcinogenesis model, lipid peroxidation and 8-OH-dG were found to increase at a very early stage, and then reached their plateau levels (34). Recently, the concept of `persistent oxidative stress in cancer' was proposed as the ROS-mediated mechanism of carcinogenesis. Persistent, rather than rapid and pronounced oxidative damage explains the characteristic biology of cancer cells (35).
An important issue that was addressed in this study was which cells at the squamocolumnar junction were the targets of oxidative damage in this model. In response to oxidative stress, cells overexpress certain genes as an adaptive or protective mechanism, such as HO1 and MT. HO1 is the highly inducible form of heme oxygenase, which catalyzes the initial and rate-limiting step in the oxidative degradation of heme to bilirubin (25). HO1 is rarely expressed in normal cells, except macrophages. When stimulated by inducers of oxidative stress, such as heme and metals, hydrogen peroxide, UV irradiation, inflammatory cytokines and lipid peroxide, many cells overexpress HO1 as a protective mechanism: it decreases the level of free cytosolic iron by inducing ferritin and increases the production of the antioxidative bile pigments (36). Knockout of the HO1 gene made the cells hypersensitive to oxidative stress induced by hemin, hydrogen peroxide, paraquat, cadmium and endotoxin, according to a recent study (37). MT is a low-molecular-weight protein with a high content of cystein residues, which detoxifies heavy metal and scavenges ROS (25). MT synthesis is known to be induced by various stresses, including heavy metals, UV irradiation, X-irradiation, producers of ROS (tert-butyl hydroperoxide, paraquat, cisplatin, etc.), glutathione consumer (diethyl maleate) and cytokines (38). Overexpression of MT protects against the cytotoxic and DNA-damaging effects induced by nitric oxide (39). Antisense downregulation of MT induced growth arrest and apoptosis in human breast cancer cells (40). Knockout of the MT gene promotes the mouse skin carcinogensis induced by 7,12-dimethylbenz[a]anthracene (41).
Normal esophagus does not express HO1 and MT. Some of the columnar epithelial cells and goblet cells of the small intestine occasionally express a low level of MT (42). In this study, we observed overexpression of both HO1 and MT in both CLE cells and EAC. This provided further evidence for the idea that the CLE cells at the squamocolumnar junction were targeted by oxidative stress. Consistent with our results, increased expression of HO1 and MT has been observed in premalignant and malignant cells of various kinds of human and animal cancers (43,44).
In conclusion, we have observed oxidative damage in the esophagus after EDA and iron supplementation. Iron overload occurred at the squamocolumnar junction not only by overexpressing TfR in the premalignant CLE cells, but also by migration of macrophages carrying `free' iron. CLE cells were the targets of oxidative stress since they were overloaded by transferrin-bound iron, surrounded by iron-overloaded macrophages, and overexpressed two oxidative stress-responsive genes, MT and HO1. We believe iron plays an important role in the formation of EAC by promoting oxidative damage in the premalignant CLE cells at squamocolumnar junction. A similar situation may occur in humans with gastroesophageal reflux and iron over-nutrition.
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Acknowledgments
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This study was supported by NIH grant CA75683 and facilities from the NIEHS Center Grant ES05022 and the Cancer Center Support Grant CA72720. C.S.Y. is a member of the Environmental and Occupational Health Sciences Institute and the Cancer Institute of New Jersey.
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Notes
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1 To whom correspondence should be addressed E-mail: csyang{at}rci.rutgers.edu

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Received June 30, 1999;
revised October 4, 1999;
accepted October 14, 1999.