Ann Thorac Surg 1998;65:779-786
© 1998 The Society of Thoracic Surgeons
Original Articles: General Thoracic
Epithelial Cell Hyperproliferation After Biliopancreatic Reflux Into the Esophagus of Rats
Manuel Pera, MD,
Luis Grande, MD,
Marisa Gelabert, MD,
Xavier Figueras, PhD,
Miguel Pera, MD,
Antonio Palacín, MD,
Montserrat Elena, MD,
Antonio Cardesa, MD,
Antonio F. Tiburcio, PhD,
Victor F. Trastek, MD
Department of Surgery, Hospital Clinic i Provincial, University of Barcelona Medical School, Barcelona, Spain
Department of Pathology, Hospital Clinic i Provincial, University of Barcelona Medical School, Barcelona, Spain,
Department of Plant Physiology, Faculty of Pharmacy, University of Barcelona, Barcelona, Spain
Dr Pera, Service of General Surgery, Hospital Clinic i Provincial, University of Barcelona Medical School, Villarroel 170, Barcelona 08036, Spain (e-mail: pera@medicina.ub.es).
Abstract presented at the Thirty-third Annual Meeting of The Society of Thoracic Surgeons, San Diego, CA, Feb 3–5, 1997.
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Abstract
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Background. Chronic reflux of duodenal contents into the esophagus of rats produces severe esophagitis and exerts a co-carcinogenic effect on the proliferating cells by enhancing the formation of nitrosamine-induced esophageal carcinomas. We investigated the effect of the different components of the duodenal reflux on the epithelial cell proliferation of the lower esophagus.
Methods. Sprague-Dawley rats underwent three surgical reflux models (biliopancreatic, pancreatic, and biliary) and a sham operation. Animals were sacrificed at 72 hours, 6 weeks, and 9 weeks after the operation. Histology and cell proliferation, determined by ornithine decarboxylase activity, polyamine (putrescine, spermidine, spermine) levels, and proliferating cell nuclear antigen labeling index of the basal and suprabasal layers, were studied in the distal esophagus.
Results. Both biliopancreatic and pancreatic reflux induced severe esophagitis starting on week 6. Suprabasal proliferating cell nuclear antigen labeling index significantly increased throughout the 9 weeks of the study in the biliopancreatic and pancreatic reflux groups, although this increase was earlier in the former group. Ornithine decarboxylase activity and polyamine levels were significantly increased in the biliopancreatic and pancreatic groups on week 6, decreasing on week 9.
Conclusions. Increased esophageal cell proliferation after both biliopancreatic and pancreatic reflux into the lower esophagus may therefore be one mechanism by which duodenal-content reflux stimulates esophageal carcinogenesis in experimental animals.
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Introduction
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Pathologic gastroesophageal reflux may expose the mucosa of the distal esophagus to both acid and duodenal-content secretions [1]. Mixed reflux to the esophagus has been implicated in the development of Barrett’s esophagus and its complications (stricture, ulceration, and dysplasia) [1][2]. In the esophagus of human subjects, epithelial cell proliferation increases in reflux esophagitis and the precancerous disease manifested by Barrett’s esophagus [3][4]. Experimental studies in rats have shown that chronic duodenal-content reflux into the esophagus induces severe esophagitis and also plays a role as a co-carcinogenic factor by increasing the number of esophageal carcinomas when a carcinogen is given simultaneously [5][6][7]. Further studies showed that reflux of pancreatic secretions alone into the esophagus was enough for the development of severe esophagitis and for the appearance of esophageal tumors when a carcinogen with strong esophagotropism was given. However, its combination with biliary secretions had a potentiating effect, increasing even more both the mucosal damage and the co-carcinogenic role [8]. It is well established that increased cell proliferation leads to an enhanced susceptibility to chemical carcinogens and, thus, to an increased tumor incidence in experimental carcinogenesis [9][10].
In tissues that have a high proliferative rate the activity of the rate-controlling enzyme in polyamine biosynthesis, ornithine decarboxylase (ODC), and the concentrations of the polyamines (spermidine and spermine and their precursor putrescine) are relatively high. Marked increases in this enzyme’s activity and rapid accumulation of cell and tissue polyamines are characteristically found after exposure to trophic stimuli. Such stimuli include hormones, drugs, tissue regeneration, and growth factors [11].
The purpose of the present study was to investigate the effect of different components of the duodenal-content reflux on epithelial cell proliferation of the lower esophagus in rats. To accomplish these goals we designed three different types of reflux models (biliopancreatic, pancreatic, and biliary) and determined several markers of cell proliferation, such as polyamine levels, ODC activity, and proliferating cell nuclear antigen labeling index (PCNA-LI).
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Material and Methods
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Animals
Male Sprague-Dawley rats weighing 200 to 250 g were housed 3 to a wire-bottomed cage and given water and standard laboratory rat food ad libitum. All animals were obtained from Iffa Credo Laboratories, France. The animals’ quarters were maintained under standard laboratory conditions. Animals were deprived of food but allowed free access to tap water for 48 hours before surgery.
The Institutional Animal Care and Use Committee of the Hospital Clinic Research Foundation at Barcelona reviewed and approved the protocol for this study. All animals received humane care in accordance with the "Guide for the Care and Use of Laboratory Animals" (NIH publication 85-23, revised 1985).
Surgical Models of Reflux
Pancreatic (P), biliary (B), and biliopancreatic (BP) reflux models were performed as previously described [8]. Sham-operated controls (S-O) underwent a midline laparotomy with manipulation of the gastroesophageal junction and distal esophagus. Anesthesia was induced and maintained with an ether-air mixture. All animals had free access to both water and food 12 and 24 hours, respectively, after the operation.
Experimental Design
The experiment was divided into two protocols. In the first protocol, 96 animals were divided into four groups (BP, P, B, and S-O) of 24 rats each. Animals from the four groups that survived the operation were then killed at 72 hours, 6 weeks, and 9 weeks after the operation. Animals were killed by an overdose of ether at a fixed time of day (between 2 and 3 PM) to avoid possible circadian rhythm of ODC. All animals had an esophageal washout during the autopsy procedure to confirm the type of reflux produced. Briefly, the esophagus was rinsed one time with 2 mL normal saline. The sample thus obtained was temporarily stored on crushed ice and later was frozen at -40°C for trypsin and bile acids determinations. The entire esophagus was removed and the lumen opened through the dorsal aspect of the esophageal wall. After washing briefly in tap water, the esophagus was pinned out on a cork board and the macroscopic findings noted. Then, the esophagus was divided into two portions, proximal and distal. Only the distal portion of the esophagus was used. It was longitudinally divided into two segments. One was assayed to determine the activity of ODC and protein content. In this segment the mucosa was scraped away from the underlying muscle. Then, mucosal scrapings were weighed and frozen at -80°C until analyzed within 3 months of sacrifice. The other segment was used for histologic study and PCNA-LI determination of the basal and suprabasal compartments of the squamous epithelium.
Because of insufficient amount of esophageal mucosa samples for simultaneous polyamine and ODC activity assessment, we decided to set up a second protocol to determine the polyamine levels. As in the first protocol, 96 animals were again divided into four groups (BP, P, B, and S-O) of 24 animals each. Animals were killed at the same times as in the first protocol. The whole distal portion of the esophagus was used for measurement of polyamines (putrescine, spermidine, and spermine) and protein content. In this segment the mucosa was scraped away from the underlying muscle. Mucosal scrapings were weighed and collected in glass tubes and immediately frozen at -80°C.
Biochemical Assays
Analysis of Bile Acids and Trypsin in Esophageal Washouts
The samples were analyzed for the presence of active trypsin and bile acids. Immunoreactive trypsin was determined by means of a classic double-antibody radioimmunoassay (Ria-gnost Trypsin, CIS Bio International, France). The within-run and between-run variability was 4.1% and 6.4% for low concentrations, 5.3% and 8.2% for normal, and 7.2% and 9.3% for above-normal concentrations. The lower limit of detection was 20 mg/L. The assay was most precise between 75 and 700 µg/L. The percentage of recovery was 104%. Conjugated bile acids were determined by means of a solid-phase, antibody-coated tube radioimmunoassay (Becton-Dickinson Immunodiagnostics, Orangeburg, NY). The within-run and between-run variability was 10% and 13%, respectively. The lower limit of detection was 0.4 µmol/L. The assay was most precise between 0.5 and 25 µmol/L.
Determination of ODC Activities
Preliminary studies showed that a minimum amount of 0.1 g of esophageal mucosa was necessary for ODC activity determination. Samples were ground in chilled mortars in a ratio of 100 mg fresh weight/mL of 100 mmol/L potassium phosphate (pH 7.5) containing 10 mmol/L dithiothreitol, 20 mmol/L sodium ascorbate, 5 mmol/L ethylenediaminetetraacetic acid, and 1 mmol/L pyridoxal phosphate. The extract was sonicated and pelleted for 20 minutes at 27,000 g. The activity of ODC was determined in the supernatant fraction as described elsewhere [12]. The labeled substrate consisted of 20 mCi/mL DL-[1-14C]ornithine (50 mCi/nmol; Amersham, Buckinghamshire, UK) diluted with unlabeled ornithine to give a final concentration of 50 mmol/L. Reaction mixtures were incubated for 45 minutes with gentle shaking at 37°C, at which time the reaction was stopped by adding 0.2 mL of 10% (v/v) perchloric acid. Trapping of the labeled CO2 onto the KOH-impregnated collection disc continued for 45 minutes at 37°C. The discs were then removed and immersed in 2 mL of Cocktail Biogreen 1 (Scharlau, Barcelona, Spain). The radioactivity liberated was determined by counting for 10 minutes in a scintillation counter. Enzymatic activity was expressed as nanomoles of 14CO2 released per milligram of protein per hour.
Mucosal Polyamine Analysis
Samples were extracted by homogenizing in 5% (v/v) cold perchloric acid at 300 mg fresh weight/mL. The homogenates were kept on ice 30 to 60 minutes before centrifugation at 27,000 g for 20 minutes. The supernatant was set aside and the pellet was resuspended in the original volume of 1 N NaOH by vortexing. The perchloric acid supernatant containing the free soluble polyamines was dansylated and chromatographed as previously described [12]. Dansyl-polyamines were separated on high-resolution silica gel thin-layer chromatography plates (Whatman LK6D, Maidstone, England). Chloroform:triethylamine (4:1, v/v) was used as a solvent. The polyamine standards putrescine, spermidine, and spermine were included each time polyamine levels were analyzed. After development of the plates, bands were scraped into 2 mL of ethyl acetate and the fluorescence at 495 nm determined in a Hitachi spectrophotofluorimeter (F-2000, Hitachi Ltd, Tokyo, Japan) with an activating wavelength of 350 nm. The amount of polyamines was expressed as nanomoles per milligram of protein.
Protein Analysis
Protein was determined according to the method of Bradford [13] in the insoluble perchloric acid pellet resuspended in 1 N NaOH. Bovine 
-globulin (Sigma, St. Louis, MO) was used as a standard. The amount of protein was expressed in milligrams.
Histology
Full-thickness segments from the esophagus were removed, fixed in 10% buffered formalin during 24 hours, and embedded in paraffin. Sections (4 µm) were stained with hematoxylin and eosin for microscopic examination. The esophagus was examined for the presence of squamous hyperplasia, papillomatosis, ulceration, and inflammation. All histologic slides were evaluated in a blinded manner by two of us (A.P. and A.C.).
PCNA-Labeling Index
To estimate esophageal cell proliferation in all animals from the four groups of the first protocol, thin slices (3 µm thick) from paraffin-embedded distal esophagi were immunostained with anticyclin/proliferative cellular nuclear antigen (PCNA) monoclonal antibody (PC-10 purchased from DAKO Corp, Carpinteria, CA) diluted 1:200. Proliferating cell nuclear antigen is an evolutionarily conserved 36-kDa nuclear protein that, by functioning as a cofactor for DNA polymerase 
, is an absolute requirement for semiconservative DNA synthesis [14]. Levels of PCNA increase dramatically during the cell cycle and it is undetectable in long-term quiescent cells. The application of antibodies to PCNA allows the possibility of objectively quantifying the number of cycling cells in tissue sections [14]. Although there was some variation in the intensity of the nuclear immunostain in different positive cells, all immunostained nuclei, independent of intensity, were scored as positive. Epithelial cell proliferation was expressed as labeling index (LI), which is defined as the percentage of PCNA-labeled nuclei per 300 epithelial cells. After dividing the epithelium into the basal and suprabasal layers, the LIs of these two compartments were determined separately as described above. The pathologists (A.P. and A.C.) performing the scoring were unaware of the animal’s characteristics.
Statistical Analysis
Data are presented as the mean ± standard error (SE) of the mean of 5 to 8 rats per group. Statistical analysis was performed using the Mann-Whitney U test for nonparametric data. The differences were considered significant at a p value of less than 0.05.
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Results
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Of the 96 animals in protocol 1, 24 were excluded (25%). Five, 2, 8, and 9 animals in the BP, P, B, and S-O groups, respectively, were not considered in the study. The causes were insufficient amount of esophageal mucosa for ODC determination in 19 cases, poorly oriented epithelium for PCNA analysis in 3 cases, intraabdominal abscess in 1, and postoperative death in 1. Of the 96 animals of protocol 2, 14 animals were excluded (14.6%). The causes were postoperative deaths in 13 cases and intraabdominal abscess in 1. Despite this high percentage of excluded animals in both study periods, a minimum of 5 rats were always available in all reflux groups at each time point of evaluation.
Trypsin and Bile Acids Determination
As shown in Table 1 the determination of active trypsin and bile acids in the esophageal washout samples confirmed the actual occurrence of each type of reflux into the esophagus in all the experimental models used and ruled out the existence of reflux in the sham-operated animals. Esophageal washouts were not performed on the second protocol group of animals because it was not felt to be necessary to demonstrate similar effective reflux as we did in the first protocol group of animals.
Morphologic Findings
Macroscopic Study
The esophageal mucosa in all animals from the B and S-O groups appeared normal at the three time periods of the study. The esophageal mucosa was also macroscopically normal in all animals from the BP and P groups that were sacrificed at 72 hours after producing each type of reflux. At 6 weeks of survival, all esophagi from animals with biliopancreatic or pancreatic reflux had dilated lower portions, at times approaching a diameter more than twice that of a normal esophagus. Extensive superficial ulcers developed on the inner surface of the lower half of the esophagus in animals with pancreatic reflux alone and biliopancreatic reflux. Also, there were longitudinal white folds intermingled with areas of ulceration. At 9 weeks of survival, the wall of the distal esophagus became more thickened, and whitish nodular patches covered the remainder of the esophageal surface giving a cobblestone appearance.
Microscopic Study
Histologic changes in the distal esophagus are shown in Table 2. The esophageal mucosa was microscopically normal in all animals with biliary reflux or a sham operation at the three time points of the study (Fig 1). At 72 hours minimal squamous hyperplasia was observed in 2 of 5 animals with biliopancreatic reflux. Animals with pancreatic reflux did not show any abnormality at this time point of evaluation. At 6 weeks microscopic examination demonstrated ulceration and fibrinoid necrosis of the internal esophageal surface in 8 and 7 animals with biliopancreatic and pancreatic reflux, respectively. The squamous epithelium, lamina propria, and muscularis mucosa were necrotic and destroyed in these areas. Generally, in the areas around the ulcer where the covering squamous epithelium was still preserved, a strong papillary hyperplasia of the squamous epithelium was observed together with the evidence of acanthosis and hyperkeratosis (Fig 2). Papillomatosis was always observed in the most distal part of the esophagus in animals with biliopancreatic or pancreatic reflux. The squamous epithelium showed reactive changes in areas close to ulcerations. At 9 weeks, the microscopic examination in both groups of animals showed more extensive ulcerations and more prominent papillomatosis. Acute and chronic inflammation was seen in the lamina propria, being minimal in the esophagus of animals with biliopancreatic reflux at 72 hours and prominent, always associated with superficial ulceration, in animals with both biliopancreatic and pancreatic reflux at 6 and 9 weeks. Columnar-lined epithelium was not observed in the distal esophagus of any of the animals studied.
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Normal esophageal squamous epithelium of rat after 6 weeks of biliary reflux in cross-section is composed of the usual basal (B), spinous (S), granular (G), and keratinized layers (K). (Hematoxylin and eosin; x400 before 41% reduction.)
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Papillary hyperplasia in an animal after 6 weeks of biliopancreatic reflux. Squamous epithelium shows thickening of the spinous layer with initial papillary elongations and prominent hyperkeratosis. The basal layer is widened. (Hematoxylin and eosin; x150 before 41% reduction.)
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PCNA-Labeling Index
Proliferating cell nuclear antigen labeling index was determined in the basal and suprabasal layers of the squamous epithelium (Fig 3Fig 4). The PCNA-LI in the basal layer shows no differences between the four groups (BP, P, B, S-O) at the three time points of evaluation, being around 80% (data not shown). The PCNA-LI in the suprabasal compartment of the squamous epithelium of the four groups is shown in Fig 5. An expansion of the proliferative compartment toward the suprabasal layer, showing increased PCNA-LI at this level, was only observed in the esophagus of animals with biliopancreatic and pancreatic reflux. It was a progressive increase during the three time periods of the study but with an earlier observation of PCNA-stained cells in the suprabasal compartment at 72 hours in the BP group. At 9 weeks suprabasal PCNA-LI in the BP and P groups reached a similar level.
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Normal esophageal epithelium from an animal after 72 hours of pancreatic reflux showing proliferating cell nuclear antigen (PCNA)-stained cells (arrows) limited to the basal layer, which is the normal proliferative compartment. (Anti-PCNA/DAB; x400 before 41% reduction.)
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Esophageal mucosa from an animal after 9 weeks of pancreatic reflux showing expansion of the proliferative compartment toward the suprabasal layer (arrows). (Anti-PCNA/DAB; x250 before 41% reduction.)
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Proliferating cell nuclear antigen (PCNA) labeling index (% of PCNA-stained cells) in the suprabasal layer after counting 300 epithelial cells at this level. Each symbolrepresents mean from at least 5 animals and barsdenote standard error of the mean. (BP = biliopancreatic reflux; P = pancreatic reflux; B = biliary reflux; S-O = sham operation; h = hours; w = weeks; ¶p < 0.05 versus P, B, S-O;  p < 0.05 versus B, S-O.)
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Esophageal Mucosal ODC Activity
As shown in Fig 6, after both biliopancreatic and pancreatic reflux there is an appreciable increase in mucosal ODC activity, occurring at week 6. Mucosal ODC activities increased by fourfold in the P group but increased 17-fold in the animals that had a biliopancreatic reflux compared with the activities observed in both the B and S-O groups. The high ODC activity decreased in both groups after week 6 and had returned toward nearly basal values by week 9. The ODC activities in animals with biliary reflux and sham operation remained low for the 9 weeks of the study.
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Activity of ornithine decarboxylase (ODC) in the esophageal mucosa at different times after three types of reflux esophagitis (BP, P, B) and a sham operation (S-O). Each symbolrepresents mean from at least 5 animals and barsdenote standard error of the mean (BP = biliopancreatic reflux; P = pancreatic reflux; B = biliary reflux; S-O = sham operation; h = hours; w = weeks; ¶p < 0.05 versus P, B, S-O; p < 0.05 versus B, S-O.)
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Esophageal Mucosal Polyamine Levels
Concurrent with the increase in mucosal ODC activity at week 6, mucosal polyamine contents also significantly increased at this time point after both biliopancreatic reflux and pancreatic reflux (Fig 7). At week 9 all three polyamines (putrescine, spermidine, and spermine) in the BP and P groups had values similar to those observed at 72 hours.
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Mucosal levels of polyamines putrescine (A), spermidine (B), and spermine (C) at different point times after three types of reflux and a sham operation. Each symbol represents mean from at least 5 animals and bars denote standard error of the mean. (BP = biliopancreatic reflux; P = pancreatic reflux; B = biliary reflux; S-O = sham operation; h = hours; w = weeks;¶p < 0.05 versus P, B, S-O; p < 0.05 versus B, S-O.)
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Comment
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The role of chronic esophageal irritation as a promotional or contributing factor of experimental esophageal carcinogenesis has been previously studied [15]. It was suggested that the healing esophagus that had been subjected to chemical or physical irritation, but which would have been in a state of enhanced epithelial regeneration, was clearly more susceptible to carcinogen-induced tumorigenesis [15]. Our group demonstrated that long-standing duodenal-content reflux (pancreatobiliary secretions) into the esophagus exerted a co-carcinogenic effect by increasing the number of carcinomas when a low-dose carcinogen was given simultaneously [5]. Other workers have also documented increased malignant degeneration in the rat cancer model in the presence of duodenal-content reflux esophagitis [6][7]. Subsequent studies have shown that the esophageal carcinoma is induced only in the presence of pancreatic secretions whereas biliary reflux appears to have a potentiating role, increasing even more the co-carcinogenic effect when in combination with pancreatic juices [8]. The mechanism of the tumor-enhancing effect remains to be clarified. Based on other experimental models, it seems established that increased cell proliferation leads to an enhanced susceptibility to chemical carcinogens and thus to an increased tumor incidence in experimental carcinogenesis [16].
The current study was undertaken to ascertain which component of the duodenal-content reflux (pancreatic, biliary, or its combination) induces higher epithelial cell proliferation in the esophageal mucosa to establish their specific contribution to the cocarcinogenic effect of the duodenal-content reflux in previous experiments [5]. Our study shows that both biliopancreatic reflux and pancreatic reflux alone induce an increase of epithelial cell proliferation in the esophageal mucosa based on PCNA-LI data. However, the combination of bile and pancreatic secretions induces an earlier increase of cell proliferation compared with pancreatic reflux alone. Bile reflux alone does not affect cell proliferation. The increased cell proliferation was documented as an expansion of the cell proliferative compartment from the basal layer toward the suprabasal layer. What causes the transfer of cells out of the basal layer? Previous studies showed that the addition of new cells by mitosis in this layer tends to increase "population pressure," being the acting factor that causes migration of these proliferating cells out of the restricted proliferation compartment [17][18]. The observation of increased numbers of PCNA-stained cells out of the basal layer is an estimate of cellular proliferation and not a marker of malignancy per se. However, malignant degeneration occurring with duodenal-content reflux in the rat model used in previous studies [5][7] was in tissues that were in a marked state of increased cell turnover. This is the ideal environment for the generation of genetic errors, a situation that may result in neoplasia. Attwood and colleagues [19] found that the presence of squamous hyperplasia on microscopy was paralleled by the increased cell turnover rates measured by flow cytometry in the same rat model. They found increased numbers of cells in the S and G2/M phases of the cell cycle. Miwa and associates [20] observed esophageal carcinomas in rats after 50 weeks of duodenal-content reflux without the administration of carcinogen. Why did the combination of pancreatic and biliary secretions exert a more potent cocarcinogenic effect than pancreatic reflux alone in our previous study [8]? The current study shows that both kinds of reflux induce the same percentage of suprabasal PCNA-LI at 9 weeks. However, it is possible that although inducing the same hyperproliferative response, the additional presence of bile might make the difference. Previous studies have demonstrated the cocarcinogenic effect of bile in colon carcinogenesis [21].
Although ODC activity and polyamine levels were determined in different esophageal samples, we observed the same peak at week 6 after biliopancreatic reflux and to a lesser degree after pancreatic reflux. The fact that a similar pattern of results was seen at every time interval (72 hours, 6 weeks, and 9 weeks) after each type of reflux also suggests that the increase in polyamine levels (putrescine, spermidine, and spermine) observed at week 6 is indeed the result of increased ODC activity. The more significant increase of ODC and polyamine levels occurring at week 6 in animals with biliopancreatic reflux compared with those with pancreatic reflux may be caused by the presence of bile. Bile acids, well-known promoters of colon carcinogenesis, cause significant induction of ODC and DNA synthesis [22]. The role of polyamine metabolism in the intestinal response to injury has been studied in several experimental models [23][24]. However, few of them were chronic models of intestinal damage as the one we have studied [25]. Surprisingly, ODC activity and polyamine levels decreased at week 9 while PCNA-LI was showing concomitant progressive expansion of the proliferative compartment. Two possible explanations may be suggested: first, the increase in ODC activity and polyamine biosynthesis might be necessary during the initial cell hyperproliferative period, favoring cell renewal and healing of the damaged squamous epithelium; second, extensive destruction of the squamous epithelium because of persistent injury as seen at week 9 may produce an overall decrease in polyamine synthesis. Future studies using ODC antibodies and ODC cDNA specific probes will help to answer this question.
In summary, this study confirms the damaging effects of duodenal-content secretions on the esophageal mucosa of rats. We have shown that pancreatobiliary secretions stimulate an expansion of the proliferative compartment of the squamous epithelium. This study may explain in part the mechanism by which duodenal-content reflux stimulates esophageal carcinogenesis in experimental animals [5][6][7]. Future studies should clarify the sequence of molecular events that develop in the hyperproliferative epithelium.
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Acknowledgments
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This study was supported by Grant 91/0249 from the Fondo de Investigacion Sanitaria, Ministry of Health, Spain.
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M. Pera, M. J. Brito, M. Pera, R. Poulsom, E. Riera, L. Grande, A. Hanby, and N. A. Wright
Duodenal-content reflux esophagitis induces the development of glandular metaplasia and adenosquamous carcinoma in rats
Carcinogenesis,
August 1, 2000;
21(8):
1587 - 1591.
[Abstract]
[Full Text]
[PDF]
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Abstract
Introduction
