REVIEW

Rat Reflux Model of Esophageal Cancer and Its Implication

in Human Disease
Christina L. Greene, MD, Stephanie G. Worrell, MD, and Tom R. DeMeester, MD

Objectives: The epidemiologic shift in esophageal cancer from squamous
cell carcinoma to esophageal adenocarcinoma coincided with popularization
of proton pump inhibitors and has focused attention on gastroesophageal
reflux disease as a causative factor in this shift. The aim of this study is to
review the literature on the rat reflux model in an effort to elucidate this
phenomenon.
Methods: An extensive online literature review (PubMed) was carried out to
identify all seminal contributions to the study of esophageal adenocarcinoma
using the rat reflux model.
Results: The rat reflux model is a validated reproducible model for the
development of Barrett’s esophagus and esophageal adenocarcinoma. Esoph-
ageal reflux of an admixture of gastric acid and duodenal juice induces Barrett’s
esophagus followed by adenocarcinoma. A high-pH environment created by
surgical gastrectomy or proton pump inhibitor therapy in combination with a
high-fat diet seems to potentiate the development of Barrett’s esophagus and
adenocarcinoma. Early surgical intervention to prevent reflux reduces the
progression toward esophageal adenocarcinoma. Anti-inflammatory, antiox-
idant, and nitrate-trapping agents reduce the incidence of tumorigenesis.
Conclusions: As in the rat so also in humans, reflux of an admixture of gastric
acid and duodenal juice in a high-pH environment induces the development of
Barrett’s esophagus followed by esophageal adenocarcinoma. This has led to
the hypothesis that to prevent Barrett’s esophagus and subsequent esophageal
adenocarcinoma in humans, the reflux of an admixture of acid and bile must
be controlled before the development of Barrett’s esophagus by methods other
than acid-suppression therapy.
Keywords: Barrett’s esophagus, cid and bile reflux, esophageal
adenocarcinoma, proton pump therapy, rat reflux model
(Ann Surg 2015;262:910–924)
I n 1972, Sir James W. Black led a team of researchers in synthesiz-
ing cimetidine, a competitive antagonist of the H2-receptor. As
planned it caused a reduction in gastric acid secretion. On premarket
testing the drug had minimal toxicity. When introduced in the United
Kingdom in 1976 and the United States in 1977, the drug showed
remarkable success in the treatment of acid peptic disease. In 1988,
Sir James Black received the Nobel Prize in Medicine for the
conception and implementation of a ‘‘rational-drug-design struc-
ture.’’ This 2-step approach to pharmaceutical development consisted
of discerning the physiological and biochemical aspects of a disease
process, and then synthesizing a pharmaceutical compound that
blocks the process. The initial outcome of this approach was the
synthesis of the H2-blocker of gastric acid secretion, cimetidine. The
subsequent discovery of the Hþ-Kþ-ATPase pump by Forte and Lee1
in 1977 led, using the concept of rational-drug-design-structure, to
From the Keck School of Medicine of the University of Southern California, Los
Angeles, CA.
Disclosure: The authors declare no conflicts of interest.
Reprints: Tom R. DeMeester, MD, Professor Emeritus, Department of Surgery,
Keck Medical Center of USC, 892 Huntington Garden Dr, San Marino, CA
91108. E-mail: Tom.DeMeester@med.usc.edu.
Copyright ß 2015 Wolters Kluwer Health, Inc. All rights reserved.
ISSN: 0003-4932/14/26105-0821
DOI: 10.1097/SLA.0000000000001207
the synthesis and marketing of the first proton pump inhibitor (PPI),
omeprazole, in 1989. Omeprazole revolutionized the treatment of
gastroesophageal reflux disease (GERD).2 It eliminated the classic
GERD symptom of heartburn by simply elevating the pH of gastric
juice with a pill. Over the next 2 decades, PPIs became one of the
most prescribed medications in America.
During the last 4 decades, a concomitant shift took place in the
epidemiology of esophageal carcinoma. Before the 1980 s, squamous
cell carcinoma was the predominant form of esophageal cancer in the
United States and adenocarcinoma of the esophagus was extremely
rare. By the mid-1980 s, esophageal adenocarcinoma accounted for
one third of all esophageal cancers in white men in the United
States,3,4 and by the turn of the century, adenocarcinoma exceeded
squamous cell carcinoma as the most common esophageal malig-
nancy. This epidemiological switch connected one of the world’s
most dreadful human cancers to GERD, a common foregut abnor-
mality. The reason for the rise in esophageal adenocarcinoma is
unclear, but its occurrence in timing with the increased use of PPIs is
undeniable.5,6 This has led to speculation that the effect of the
medication on the gastric pH and in turn on the chemistry of bile
acids may be linked to the rise in adenocarcinoma of the esophagus.7
The aim of this study is to review the current literature on the
rat reflux model to determine the tumorigenic effect of refluxed
gastric juice, composed of acid and bile at various pH, on the
esophageal mucosa and to elucidate how this effect may have
contributed to the switch in tumor histology. For conformity, the
term ‘‘Barrett’s’’ is only used to express the existence of a mucosa
made up of columnar epithelium with goblet cells.
METHODS
An extensive online literature review (PubMed) was carried
out to identify all seminal contributions to the study of esophageal
adenocarcinoma using the rat reflux model.
RESULTS
Transition From Squamous Cell Carcinoma to
Adenocarcinoma
The first rat model for esophageal squamous cell carcinoma
was developed in 1979 by Bulay and Mirvish.8 They injected rats
with a known carcinogen, methyl-n-amylnitrosamine (MNAN), at
varying concentrations over a 6- to 12-week period, and were able to
induce squamous cell carcinoma in 40% to 65% of the rats. The
model proved to be extremely reliable and predictable in inducing
squamous cell carcinoma.
In 1989, nearly a decade later and after mounting clinical
evidence suggested a relationship between esophageal adenocarci-
noma and chronic reflux esophagitis, Pera and colleagues9 were able
to induce esophageal adenocarcinoma in rats. Adenocarcinoma
developed in 24% to 38% of rats, with surgically induced chronic
duodenogastroesophageal reflux and exposure to a known carcino-
gen 2,6-dimethylnitrosomorpholine. The surgical procedure used to
induce chronic esophageal reflux was the Levrat rat reflux model,10
an end-to-side esophagojejunostomy with gastric preservation
(Fig. 1). Adenocarcinoma developed only in rats with surgically

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Annals of Surgery  Volume 262, Number 6, December 2015
FIGURE 1. Levrat model: an esophagojejunostomy with gastric
preservation.9,10 The model became the most common rat
reflux model used.
induced reflux esophagitis, which underscores the importance of
an initial inflammatory process. The authors also reported that
the initiation of surgically induced reflux esophagitis before the
exposure of the carcinogen was associated with an increase in the
number of animals that developed cancers, shortened the tumor
induction time, and concentrated the location of the adenocarcino-
mas in the middle and distal esophagus.
Effect of Duodenoesophageal Reflux on the
Development of Esophageal Adenocarcinoma With
Carcinogen
In 1992, Attwood and colleagues11 studied the carcinogenic
effect of gastroesophageal reflux by performing an esophagogas-
troplasty to induce gastroesophageal reflux in one group of rats
and a modification of the rat reflux model to induce duodenogas-
troesophageal reflux in a second group of rats. A third group
of rats, which did not undergo surgery, made up the control group.
All of the rats in the 3 groups were exposed to the carcinogen 2,6-
FIGURE 2. Surgical operations used
by Attwood and colleagues11 to study
esophageal exposure to acid and both
acid and bile. Gastroesophageal reflux
was induced by an esophagogastroplasty
and duodenogastroesophageal reflux
was induced by a modification of the
rat reflux model to a side-to-side and
an end-to-side esophagoduodenostomy.
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Animal Models of Esophageal Cancer
dimethylnitrosomorpholine. Compared with the control group, the
rats with duodenogastroesophageal reflux doubled the frequency of
malignant tumors and altered their differentiation from predomi-
nantly squamous to 50% glandular carcinoma. This phenomenon did
not occur with gastroesophageal reflux alone, implying that the
duodenal juice, not the gastric juice, contained the necessary ingre-
dient for the development of esophageal adenocarcinoma.
Although it was becoming clear that the reflux of duodenal
juice into the esophagus promoted the tumorigenesis of esophageal
adenocarcinoma, it remained unknown what role gastric acid had in
the process. Attwood had observed limited tumorigenesis and no
adenocarcinomas with the reflux of gastric acid alone. To investigate
the role of gastric acid in the tumorigenesis of esophageal adeno-
carcinoma, Ireland and colleagues12 performed 5 different operations
on 200 rats. Twenty of the rats were nonoperative controls, whereas
the remainder underwent 1 of 5 operations consisting of a modifi-
cation of the rat reflux model, an esophagoduodenostomy, with
various degrees of gastrectomy. The operations were designed to
reflux duodenal juice mixed with various percentages of gastric juice
into the esophagus (Fig. 2). After the surgical procedure, all rats were
exposed to the carcinogen (methyl-n-amyl nitrosamine).
The authors reported a progressive increase in the prevalence
of adenocarcinoma when the esophagus was exposed to lesser and
lesser amounts of gastric acid mixed with duodenal juice (Fig. 3A).
When the esophagus was exposed to duodenogastric reflux from a
preserved intact stomach, only 30% of rats developed esophageal
adenocarcinoma. In contrast, 87% of the rats with a complete
gastrectomy developed esophageal adenocarcinoma when the esoph-
agus was exposed to pure duodenal juice without gastric acid. Of
importance, the various procedures had no effect on the incidence of
squamous cell carcinoma (Fig. 3B). The authors concluded that the
distal esophagus was the preferred location for the emergence
of esophageal adenocarcinoma and that duodenogastroesophageal
reflux potentiates the development of esophageal cancers, nearly half
of which are adenocarcinomas. Changes in the admixture of gastric
acid with duodenal juice correlated indirectly with the degree of
tumorigenesis. The presence of gastric acid seemed to suppress the
development of adenocarcinoma. The authors expressed the concern
that the popular use of continuous profound acid-suppression therapy
in the form of PPIs could be harmful, especially in patients who
reflux an admixture of gastric and duodenal juice into their esoph-
agus. They suggested it may be more prudent to stop the reflux of
such an admixture into the esophagus with an antireflux procedure
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Greene et al
FIGURE 3. Modification of the rates of esophageal adenocar-
cinoma (A) and the rates of squamous cell carcinoma (B) by
extent of gastric retention. Prevalence of esophageal adeno-
carcinoma is higher when the esophagus is exposed to smaller
amounts of gastric juice caused by a greater degree of gastric
resection, suggesting gastric juice has a protective effect.
Presence of gastric juice had no effect on the prevalence
of squamous cell carcinoma of the esophagus. ED indicates
esophagoduodenostomy; EDAR, esophagoduodenostomy
with proximal gastrectomy and antral retention; EDB1, esoph-
agoduodenostomy and antrectomy with Billroth I reconstruc-
tion; TGED, total gastrectomy with esophagoduodenostomy.12
until further studies could be performed on the safety of continuous
acid-suppression therapy.
Effect of Duodenoesophageal Reflux on the
Development of Esophageal Adenocarcinoma
Without Carcinogen
Building on the concept that duodenal juice contained an
adenocarcinoma-producing carcinogen, Clark et al13 explored the
effect of dietary fat on the development of Barrett’s esophagus and
adenocarcinoma with and without the use of a carcinogen. The
modified rat reflux model with preservation of the stomach
(Fig. 4) was used to expose the lower esophagus to gastroduodenal
juice. Despite the absence of the carcinogen, nitrosamine MNAN,
rats developed esophagitis, metaplastic Barrett’s esophagus, dys-
plastic Barrett’s esophagus, and unexpected squamous cell carci-
noma of the esophagus. No adenocarcinomas were observed. A high-
fat diet had no effect on the incidence of metaplasia or neoplasia
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Annals of Surgery  Volume 262, Number 6, December 2015
FIGURE 4. Esophagoduodenostomy with gastric preser-
vation.13 This became the most common modification of the
Levrat rat reflux model to induce duodenogastroesophageal
reflux.
(Table 1). The serendipitous discovery of a squamous cell carcinoma
was the first indication that exposure of the lower esophagus to
gastroduodenal juice could induce a malignancy without the use of
a carcinogen.
The effects on rats exposed to the carcinogen are shown in
Table 2. In the control rats without an esophagoduodenostomy, no
tumors were observed even though they received the carcinogen. In
the rats with an esophagoduodenostomy, the carcinogen increased
the occurrence of Barrett’s metaplasia and squamous cell carcinoma,
and induced adenocarcinoma. The highest incidence of adenocarci-
noma was found in rats fed a high-fat diet. Statistically, rats fed a
high-fat diet had more tumorigenesis compared with all other rats.
The investigators concluded that reflux of duodenogastric
juice without a carcinogen induced both metaplastic and dysplastic
Barrett’s esophagi. The injection of a carcinogen accelerated the
induction of adenocarcinoma, and the ingestion of a high-fat diet
caused a further increase in tumorigenesis.
A limitation of studies up to this time was that the carcinogen
was injected in addition to the reflux of duodenal juice. In the human
scenario of chronic GERD complicated by esophageal adenocarci-
noma, patients are not knowingly exposed to a carcinogen. Con-
sequently, the applicability of these studies to the real-world of
medicine was limited. This prompted Fein and colleagues14 to
investigate the effect of refluxed duodenal juice on rat esophagi
without exposure to a carcinogen. They set out to confirm that
duodenoesophageal reflux could cause esophageal adenocarcinoma
without the use of an external carcinogen. They were also intrigued
by the hypothesis of Ireland et al12 that a possible ingredient in
gastric juice could suppress the tumorigenesis of esophageal adeno-
carcinoma, and hypothesized that the protective factor was the acidic
pH of the gastric juice.
To investigate the first question, 3 groups of 26 rats were
randomized to 1 of the 3 operations using variations of the rat reflux
model: an esophagojejunostomy to induce duodenogastroesophageal
reflux, an esophagojejunostomy plus a total gastrectomy to induce
pure duodenoesophageal reflux, or a Roux-en-Y esophagojejunos-
tomy to eliminate gastric and duodenal reflux (Fig. 5). After 16
weeks, the rats were sacrificed. Adenocarcinoma occurred in 55% of
rats with duodenogastroesophageal reflux, 42% of rats with pure
duodenoesophageal reflux, and 0% of rats with a Roux-en-Y limb
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Annals of Surgery  Volume 262, Number 6, December 2015 Animal Models of Esophageal Cancer

TABLE 1. Esophageal Characteristics in Rats Not Receiving MNAN

Operation þ Operation
No Operation þ Operation þ Control Diet þ High-fat Diet
Chow (n ¼ 10) Chow (n ¼ 13) (n ¼ 14) (n ¼ 12)
Esophageal length, cm 8.6  0.6 7.1  0.7y 7.3  0.6y 6.6  0.7y
Maximal esophageal width, cm§ 0.5  0.1 1.1  0.2ô 1.1  0.2ô 1.2  0.2ô
Diffuse papillomatosis 0 13jj 14jj 11jj
Columnar lining (metaplastic BE) 0 1 0 3
Dysplasia (dysplastic BE) 0 0 1 2
Papilloma 0 1 0 1
Cancer 0 0 0 1

Significant difference between the groups, P < 0.01, ANOVA, F value 16.0.
ySignificant difference versus nonoperated controls, P < 0.05, with multiple t tests corrected with the Bonferroni technique.
zSignificant difference versus operated rats fed semipurified control diet, P < 0.05, with t test correct with the Bonferroni technique.
§Significant difference between the groups, P < 0.01, ANOVA, F value 28.3.
ôSignificant difference versus nonoperated controls, P < 0.05, with multiple t tests corrected with the Bonferroni technique.
jjSignificant difference versus nonoperated controls, P < 0.01, Fisher exact test.
BE indicates Barrett’s esophagus.

TABLE 2. Esophageal Characteristics in Rats Receiving MNAN

Operation þ Operation þ Operation þ
No Operation þ Operation þ Calorie-restricted Control Diet High-fat Diet
Chow (n ¼ 15) Chow (n ¼ 26) Diet (n ¼ 27) (n ¼ 29) (n ¼ 29)
Esophageal length, cm 8.3  0.6 6.3  0.7y 6.6  0.6y 6.6  0.6y 6.3  0.6y
Maximal esophageal width, cmz 0.5  0.1 1.1  0.2y 1.0  0.2y 1.1  0.2y 1.1  0.3y
Diffuse papillomatosis 0 25§ 27§ 29§ 29§
Columnar lining (metaplastic BE) 0 7ô 0 4 3
Dysplasia (dysplastic BE) 0 23§ 17§ 22§ 22§
Papilloma 2 5 6 10 12
Cancer 0 11§ 15§ 13§ 24§jj

Significant difference between groups, P < 0.01, ANOVA, F value 31.0.
ySignificant difference versus nonoperated controls, P < 0.05, with multiple t tests corrected with the Bonferroni technique.
zSignificant difference between the groups, P < 0.01, ANOVA, F value 32.9.
§Significant difference between versus nonoperated controls, P < 0.01, Fisher exact test.
ôSignificant difference versus nonoperated controls, and operated rats fed calorie-restricted diet, P < 0.05, Fisher exact test.
jjSignificant difference compared with all operated groups, P < 0.05, Fisher exact test.
BE indicates Barrett’s esophagus.

FIGURE 5. Modification of the rat reflux

model used to show that duodenoeso-
phageal reflux induces esophageal
adenocarcinoma without exogenous car-
cinogen. Gastroduodenal reflux: esoph-
agojejunostomy (EJ); duodenal reflux:
esophagojejunostomy plus total gastrec-
tomy (EJ-TG); no reflux: Roux-en-Y
esophagojejunostomy.14 The length of
the Roux limb was varied between 15
and 22 cm to determine the length that
produced effective bile diversion.

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Greene et al
greater than 22 cm. The authors concluded that duodenogastroeso-
phageal reflux by itself is carcinogenic in the rat reflux model and an
exogenous carcinogen is not necessary for cancer development.
Furthermore, the carcinogen must be present or develop in the
duodenal juice.
To address the second question regarding the protective effect
of acidic gastric juice, 42 rats underwent esophagojejunostomy with
total gastrectomy to produce pure duodenoesophageal reflux. One
week after surgery, the rats were randomized to receive tap water or
acidified water with a pH of 1.8. Evidence of a columnar-lined
esophagus occurred in 87% of all rats, and nearly half developed
adenocarcinomas at 16 weeks without the carcinogen. Although
there was a trend toward less adenocarcinoma if the rats received
acidified water, it was not statistically significant. The authors
concluded that the pH of the gastric juice might suppress tumori-
genesis, but further investigation was necessary to confirm this
finding. They hypothesized that the pH environment at the anasto-
mosis may be relevant in tumorigenesis. Their thought was based on
the knowledge that environmental pH was known to modify the
precipitation, solubility, and dissociation of bile acids and encourage
the formation of nitroso-bile-acid conjugates, some of which were
shown to be potential direct-acting mutagens or carcinogens.15
Effect of pH Environment on the Induction of
Esophageal Adenocarcinoma by the Reflux of
Duodenogastric Juice
The studies of Ireland et al and Fein et al suggested that
tumorigenesis of esophageal adenocarcinoma was affected by the pH
of the refluxed gastric juice. To elucidate the role of pH in the
process, Cheng and colleagues investigated the effect of environ-
mental pH on the tumorigenesis of duodenogastroesophageal
reflux.16 They studied the development of esophageal adenocarci-
noma in rats exposed to duodenogastric juice of different pH by
placing the duodenoesophageal anastomosis of the modified rat
reflux model at various distances distal to the pylorus (Fig. 6A).
After recovery from surgery, the luminal pH of the foregut was
measured (Fig. 6B). The study consisted of 3 groups of 30 rats: a low-
pH group with an anastomosis 1 cm distal to the pylorus, a high-pH
group with an anastomosis 2 cm distal to the pylorus, and a sham-
operated group as a control. Forty weeks after surgery, the incidence
of esophagitis, nondysplastic Barrett’s esophagus, dysplastic Barrett’s
esophagus, and esophageal adenocarcinoma was assessed. There was
no difference in the incidence of esophagitis or nondysplastic Barrett’s
FIGURE 6. Effects of pH on duodenogastroesophageal reflux-induced esophageal disease and cancer. A, An illustration of the
position of the ED performed in ED-A and ED-B.16 B, pH in the esophagus, fore-stomach, remaining stomach (opsthogaster), and
duodenum before and after the construction of ED-A and ED-B. Note change in pH of the distal esophagus by making a 1-cm
adjustment in the location of the esophagoduodenostomy. ED indicates esophagoduodenostomy.16
914 | www.annalsofsurgery.com
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Annals of Surgery  Volume 262, Number 6, December 2015
esophagus between the low and high-pH groups. In contrast, rats in
the high-pH group had a higher incidence of dysplastic Barrett’s
esophagus (52% vs 20%; P ¼ 0.02) and esophageal adenocarcinoma
(39% vs 8%; P ¼ 0.01) than rats in the low-pH group. The authors
concluded that duodenogastroesophageal reflux in a high-pH
environment increases the occurrence of Barrett’s esophagus with
dysplasia and esophageal adenocarcinoma, whereas a low-pH
environment has a protective effect against dysplasia and tumori-
genesis. They suggested that control of the duodenal component
and pH of refluxed gastric juice may be important factors in the
prevention of esophageal adenocarcinoma.
Effect of Dietary Fat on the Bile Acid Composition
of Refluxed Duodenogastric Juice and Its Impact via
CDx2 on the Development of Barrett’s Esophagus
and Esophageal Adenocarcinoma
Acceptance of the concept that reflux of duodenal contents
into the esophagus could initiate an esophageal adenocarcinoma, and
that the process was sensitive to pH, stimulated the study of bile acid
composition in the refluxed duodenal juice. Chen and colleagues
investigated whether there was a specific alteration in bile acid
composition that was more tumorigenic.17 They evaluated the bile
acid composition of the duodenal juice in unoperated rats fed diets
varying in the amount of cow fat versus soybean fat. They found no
significant differences in the total bile acid levels between the groups
but did observe that a high-cow-fat diet significantly increased the
concentration of taurocholic acid, a taurine-conjugated bile acid,
in the refluxed duodenal juice compared with rats fed the low-
soybean-oil diet (P < 0.05).
In a follow-up study, the investigators used the Levrat rat
reflux model (Fig. 1) to induce duodenogastroesophageal reflux in
rats fed a high-cow-fat diet or a low-soybean-oil diet.17 A similar
number of rats in both groups were killed at postoperative weeks 10,
20, and 30. At 10 weeks after surgery, erosive esophagitis and
ulceration were present in both groups. At 20 and 30 weeks after
surgery, the incidence of Barrett’s esophagus and Barrett’s dysplasia
was significantly higher in the rats fed a high-cow-fat diet (100% and
79%, respectively, vs 50% and 13%, respectively; P < 0.05). The
incidence of esophageal adenocarcinoma was also higher in rats fed a
high-cow-fat diet (43%) than in those fed a low-soybean-oil diet
(13%). Tumors in the rats fed a high-cow-fat diet were more
advanced with one third of them extending into the adventitia,
one of which extended into the liver.
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Annals of Surgery  Volume 262, Number 6, December 2015
The authors concluded that a diet high in animal fat can increase
the concentration of taurine-conjugated bile acids in the refluxed
duodenal juice and promote the development of Barrett’s esophagus
and Barrett’s dysplasia leading to esophageal adenocarcinoma. They
hypothesized that the gastric pH of rats with intact stomachs was less
than 4. This allows unionized taurine-conjugated bile acids, which are
more prevalent in animal fat diets, to penetrate the epithelial cell wall
and induce the expression of Cdx2, a gene that encodes for an
intestine-specific transcription factor involved in cell turnover and
intestinal differentiation. Cdx2 is known to play an important role in
the induction and maintenance of Barrett’s esophagus.18–20
To further elucidate the interaction between duodenogastroe-
sophageal reflux, Cdx2 and Barrett’s esophagus, Ingravallo and
colleagues21 used a modified rat reflux model to induce duodeno-
gastroesophageal reflux and study the expression of Cdx2 protein by
immunohistochemistry over the whole spectrum of the phenotypic
changes. Seventy-four rats had a modified rat reflux model con-
structed by performing a side-to-side esophagogastrojejunostomy at
the gastroesophageal junction with gastric preservation to induce
duodenogastroesophageal reflux. The rats were killed at various
times after the surgical procedure: an ‘‘early’’ group at less than
10 weeks, a ‘‘middle’’ group at 10 to 30 weeks, and a ‘‘late’’ group at
more than 30 weeks. No Cdx2 expression was detected in either the
squamous epithelia of the proximal esophagus or the 4 squamous cell
carcinomas that developed. De novo Cdx2 expression was docu-
mented consistently in the proliferative zone of the squamous
epithelium close to reflux ulcers, in the areas of multilayered
epithelium, in the areas of Barrett’s esophagus, and in esophageal
adenocarcinomas. Over the course of the experiment, an increasing
overall Cdx2 expression was documented (P ¼ 0.001). The authors
concluded that de novo expression of Cdx2 is an early event in the
spectrum of histologic changes induced by duodenogastroesophageal
reflux and is a key step in the morphogenesis of Barrett’s esophagus
and esophageal adenocarcinoma.
In addition to the stimulation of Cdx2 expression, bile acids in
the refluxed gastric juice have subsequently been shown in esoph-
ageal adenocarcinoma cells to activate genes involved in cell signal-
ing pathways, inflammation, and neoplasia. Included in these genes
is NF-kB, a transcription nuclear factor that resides inactive in the
cytoplasm of the cell bound to its inhibitor protein IkB. Upon
activation, IkB is degraded leaving NF-kB free to react with the
nucleus where it induces the transcription of its target genes, many of
which are linked to the 6 hallmarks of neoplasia,12,13,22 namely,
stimulating cell proliferation, evading apoptosis14–16,22 and promot-
ing angiogenesis, invasion, and metastasis.17– 19,22 In addition, NF-
kB plays a pivotal role in the formation of Barrett’s esophagus by
inducing the proinflammatory cytokine, interleukin-8 (IL-8).20,22
The activity level of NF-kB has been shown to increase with the
phenotypic progression of reflux esophagitis to Barrett’s esophagus
and to esophageal adenocarcinoma.21– 23
Validation That the Rat Reflux Model Produces
True Esophageal Intestinal Metaplasia
While validity of the rat reflux model was becoming more
established, there were lingering concerns by investigators that the
replacement of esophageal squamous mucosa by intestinal-type
columnar mucosa was not due to the exposure of squamous
epithelium to refluxed duodenogastric juice but rather due to the
migration of the adjacent jejunal mucosa up into the esophagus.
To validate the model, Oh and colleagues23 quantified the gene
expression of trefoil factor genes in the newly acquired esophageal
columnar mucosa and compared it with the adjacent mucosa in the
jejunum to identify the etiology of the newly acquired esophageal
columnar mucosa.
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Animal Models of Esophageal Cancer
Duodenogastroesophageal reflux was induced in 25 rats using
the Levrat rat reflux model (Fig. 1), and the animals were killed at
16 and 30 weeks. Columnar mucosa was present in the esophagus
proximal to the anastomosis in all animals, and the extent
of columnar replacement was greater after 30 weeks than after
16 weeks (Fig. 7). Goblet cells were identified within the esophageal
columnar mucosa in 50% of the rats at 16 weeks and in 100% of the
rats at 30 weeks. Invasive cancer occurred within the columnar-lined
segment in 20% of the rats. RNAs from the intestinalized columnar
mucosa in the distal esophagus, the normal jejunum, and the normal
colon were obtained and polymerase chain reaction was (PCR)
performed. Gene expression of the intestinalized columnar mucosa
in the esophagus differed significantly from the normal jejunal
mucosa and was a true metaplastic epithelium similar to human
Barrett’s esophagus. The investigators concluded that esophageal
intestinal mucosa induced by duodenogastroesophageal reflux was
true intestinal metaplasia and not proximal migration of the jejunal
mucosa. This validated the rat reflux model as a reliable model for
studies on the pathogenesis of Barrett’s esophagus and esophageal
adenocarcinoma.
Reflux of Gastric and Duodenal Contents Causes
Mutations in Esophageal Mucosa
The accumulative evidence from the above studies strongly
supported the conclusion that in the rat, duodenogastroesophageal
reflux was linked to the development of Barrett’s esophagus and the
induction of esophageal adenocarcinoma. Despite these obser-
vations, evidence was lacking that the reflux of duodenogastric juice
could induce mutations in the esophageal mucosa that are common to
neoplastic processes. To test the mutagenic potential of duodeno-
gastric juice, Theisen and colleagues24 used the modified rat reflux
model in commercially available transgenic rats, which allowed the
measurement of tissue-specific mutation frequency. Seven transgenic
rats had a modification of the rat reflux model, an esophagoduode-
nostomy, to establish duodenogastroesophageal reflux, and 14 trans-
genic rats served as nonoperative controls. Half of the control rats
FIGURE 7. Photomicrograph of the distal esophagojejunal
anastomosis. The location of the anastomosis is identified by
the dots made by embedded sutures and connected by the
black line. Columnar mucosa with goblet cells lines the distal
esophagus with a distinct transition to jejunal mucosa after the
anastomosis.23
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Greene et al
were positive controls and received intraperitoneal injections of the
carcinogen methyl-n-amyl-nitrosamine, and the other half did not
receive the injection of a carcinogen and were negative controls
(Fig. 8A). All of the rats with esophagoduodenostomy developed
gross esophagitis. Duodenogastroesophageal reflux induced a
mutation frequency 1.5-fold greater in comparison to the negative
control rats that did not receive the carcinogen (P < 0.007). The
positive control rats that received the carcinogen had a 10-fold
increase in mutation frequency in comparison to the experimental
rats with duodenogastroesophageal reflux (Fig. 8B). One of the most
striking observations of this study was that the nucleotide trans-
versions were similar to the p53 mutation patterns seen in human
esophageal adenocarcinoma. This study provided the first genetic
evidence that duodenogastroesophageal reflux can induce mutations
in the esophageal mucosa that are commonly found in human
gastrointestinal adenocarcinomas.
FIGURE 8. A, Study groups to evaluate the mutagenic poten-
tial of duodenogastroesophageal reflux: (1) Ctrl ¼ Unoperated
control rats. (2) MNAN ¼ Unoperated positive controls rats
injected with an intraperitoneal mitogen for 10 weeks. (3)
ED ¼ Rats that had duodenogastroesophageal reflux induced
by the modified rat reflux model: an esophagoduodenos-
tomy.24 B, Mutation frequency in the positive and negative
control groups and experimental group. The black bar
indicates the median value.
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Annals of Surgery  Volume 262, Number 6, December 2015
Validation That the Rat Reflux Model of Esophageal
Adenocarcinoma Meets the Criteria of Cancer
Before esophageal adenocarcinoma produced in the rat reflux
model could be accepted as a malignancy, the tumors produced
needed to demonstrate a neoplastic potential. To systematically
characterize the neoplastic potential of tumors produced in the Levrat
rat reflux model, Bonde and colleagues25 induced duodenogastroe-
sophageal reflux in 12 rats (Fig. 1). All animals developed segments
of Barrett’s mucosa consisting of columnar epithelium with intes-
tinal-type goblet cells. Nearly three fourths of the rats developed
esophageal tumors: 4 adenocarcinomas, 2 squamous cell carcinomas,
and 3 adenosquamous cell carcinomas. All of the tumors developed
within the Barrett’s mucosa. After 9 months, 3 cell culture lines were
established from 2 of the tumors. Each of the cell lines was capable of
sustained in-vitro propagation and grew as xenografts in both
subcutaneous tissue and the tissue surrounding their orthotopic
location at the gastroesophageal junction, thus confirming their
malignancy.25 Furthermore, the cytology and gene expression pro-
files of the cell lines compared favorably with normal esophageal
mucosa, and karyotype analysis showed cytogenetic heterogeneity,
aneuploidy, and a high percentage of tetraploid chromosomes.
Nearly all cell lines had loss of the Y chromosome, a genetic
mutation known to be very common in human Barrett’s esophagus
and esophageal adenocarcinoma. In addition, extensive PCR analysis
identified numerous transcripts overexpressed in rat Barrett’s epi-
thelium (VEGF, PLK, CDK4, HIF1a, and IGF-1), which have been
shown to be important in human esophageal adenocarcinoma.
The investigators summarized their findings as follows: (1)
The rat reflux model was capable of inducing metaplastic epithelial
changes simulating Barrett’s esophagus with subsequent neoplastic
transformation at a high frequency. (2) The tumors formed were
capable of continuous in vitro and in vivo propagation, thus validat-
ing their neoplastic nature. The investigators concluded that the rat
reflux model is useful to study the genesis and impact of various
therapies on the natural history of Barrett’s esophagus and esoph-
ageal adenocarcinoma.
The neoplastic potential of tumors produced by the rat reflux
model was further confirmed by Gibson et al26 who studied the
activated hedgehog (Hh) pathway in rats with gastroduodenoeso-
phageal reflux. Although the investigators were concerned with
blocking the Hh pathway with the smoothened (Smo) inhibitor to
prevent the development of Barrett’s esophagus and esophageal
adenocarcinoma, they incidentally demonstrated metastases to the
liver and lung during their detailed necropsies, confirming the neo-
plastic potential of the model.26–28 It is expected that a focused
publication of metastatic lesions in the rat reflux model will be
forthcoming from this group.
Effect of Acid-suppression Therapy and Surgical
Therapy on Evolution of Barrett’s Esophagus and
Esophageal Adenocarcinoma Using the Rat Reflux
Model
By 2012, studies with the rat reflux model had established and
validated that esophageal adenocarcinoma was induced by duodeno-
gastroesophageal reflux. The investigations by Ireland et al,12 Chen
et al,17 and Cheng et al16 gave birth to the hypothesis that an elevated pH
environment in the presence of duodenoesophageal or duodenogastroe-
sophageal reflux promoted the development of nondysplastic Barrett’s
esophagus, dysplastic Barrett’s esophagus, and esophageal adenocarci-
noma, whereas a low-pH environment exerted a protective effect. These
findings encouraged the use of the rat reflux model to study the effects of
environment pH, caused by PPI therapy, on the development of Barrett’s
esophagus and esophageal adenocarcinoma.
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Annals of Surgery  Volume 262, Number 6, December 2015
In 2012, Nasr and colleagues29 using the modified rat reflux
model compared the effect of duodenoesophageal reflux on the
development of Barrett’s esophagus when acid was present,
decreased by PPI or totally absent.29 Rats were randomized to 4
groups: (1) a ‘‘control group,’’ (2) a ‘‘combined reflux’’ group
consisting of rats that underwent esophagoduodenostomy to induce
duodenogastroesophageal reflux, (3) a ‘‘PPI treatment group’’ con-
sisting of rats that underwent esophagoduodenostomy to induce
duodenogastroesophageal reflux and were treated with PPI therapy,
and (4) a ‘‘gastrectomy’’ group consisting of rats that underwent
esophagoduodenostomy and a total gastrectomy to eliminate all acid
and induce pure non-acid duodenoesophageal reflux (Fig. 9).
Animals were deliberately killed at 26 weeks to allow observation
of the early histologic changes in the esophageal mucosa. The
incidence of Barrett’s esophagus was greater when acid was sup-
pressed by PPI therapy or gastrectomy than when not suppressed
(62% vs 27%, P ¼ 0.004, and 71% vs. 27%, P < 0.001, respectively)
(Table 3). An interesting observation was that PPIs eliminated acute
esophageal inflammation, but unexpectedly had no effect on chronic
esophageal inflammation. In addition, the tumor marker epidermal
growth factor receptor was elevated in the PPI group and the markers
Ki-67 and proliferating cell nuclear antigen were elevated in the
gastrectomy group when compared with the combined reflux group.
The investigators concluded that in the presence of duodenogastroe-
sophageal reflux, acid-suppression therapy by medical or surgical
means increased the occurrence of Barrett’s esophagus and poten-
tially increased the risk of esophageal adenocarcinoma.
Nishijima and colleagues30 used the rat reflux model to
determine whether the surgical elimination of bile reflux would
reduce the risk of developing esophageal adenocarcinoma. Two
hundred rats were randomized to 3 groups (Fig. 10). The rats in
the first group underwent an esophagojejunostomy and a total
gastrectomy to induce pure nonacidic duodenoesophageal reflux
and were killed at 20, 30, or 50 weeks. The rats in the second group
comprised the control group and underwent a total gastrectomy and
Roux-en-Y esophagojejunostomy to eliminate all reflux of either
acid or bile and were killed at 50 weeks. The rats in the third group
FIGURE 9. Surgical procedure performed on the 4 groups of rats to study the incidence of Barrett’s esophagus resulting from
various mixtures of acid and bile reflux into the esophagus. A, Control group: decreased acid and bile reflux. B, Combined reflux
group: increased acid and bile reflux. C, PPI therapy group: decreased acid and increased bile reflux. D, Gastrectomy group:
decreased acid and increased bile reflux.29
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Animal Models of Esophageal Cancer
underwent an esophagojejunostomy and a total gastrectomy to
induce pure nonacidic duodenoesophageal reflux. At 20 or 30 weeks
after their initial operation, the rats were converted to a Roux-en-Y
esophagojejunostomy to eliminate all bile reflux in addition to the
elimination of acid reflux by the initial gastrectomy (ie, the complete
elimination of all reflux), and were killed at 50 weeks.
Barrett’s esophagus occurred in 54% of rats exposed to pure
nonacid duodenoesophageal reflux for 20 weeks, 92% for 30 weeks,
and 100% for 50 weeks (Fig. 10). The incidence of adenocarcinoma
was lowest in rats exposed for 20 weeks (8%) and highest when
exposed for 50 weeks (60%) (Fig. 10). The 60% incidence of
adenocarcinoma at 50 weeks was reduced to 19% when biliary
diversion was performed at 30 weeks and to 3% when performed
at 20 weeks (P < 0.005). These incidences were similar to the
incidence of adenocarcinoma in the first group of rats killed at
20 weeks, higher than those killed at 30 weeks, and lower than those
killed at 50 weeks (Fig. 10). Carcinoma did not occur in the control
group. The investigators concluded the following: (1) Barrett’s
esophagus and esophageal adenocarcinoma can be induced by
duodenoesophageal reflux. (2) Biliary diversion performed after
the establishment of Barrett’s esophagus does not lead to regression
of Barrett’s esophagus or the elimination of the risk of esophageal
adenocarcinoma but seems to reduce progression toward esophageal
adenocarcinoma. The authors suggested that performing antireflux
surgery in patients with Barrett’s esophagus to stop duodenoesopha-
geal reflux is unlikely to eliminate but may reduce the risk of
subsequent esophageal adenocarcinoma. If performed before the
development of Barrett’s esophagus, the risk of esophageal adeno-
carcinoma is eliminated. They stressed however that the extrapol-
ation of data from animal models to humans must be done with
great caution.
Koak and colleagues31 were the only investigators identified
who used the rat reflux model to compare the effect of treating
duodenogastroesophageal reflux surgically with a Roux-en-Y esoph-
agojejunostomy versus medically with PPI therapy. Thirty rats
underwent a vagal preserving esophagojejunostomy to induce
duodenogastroesophageal reflux. Twelve weeks after the initial
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Greene et al Annals of Surgery  Volume 262, Number 6, December 2015

TABLE 3. Histology of the Distal Esophagus

Study Group n Inflammation Acute Inflammation Chronic Barrett’s Metaplasia
Control 33 0% 9% 0%
Combined reflux (no treatment) 34 94% 97% 27%
PPI therapy 34 8%y 100% 62%z
Gastrectomy 35 100% 100% 71%§

All animals were killed at 26 weeks postoperatively.
yCompared with control (P ¼ NS).
zCompared with combined reflux (P ¼ 0.004).
§Compared with combined reflux (P < 0.001).

FIGURE 10. Impact of the biliary diversion procedure on carcinogenesis of Barrett’s esophagus in the rat. The operations performed
in the 3 groups of rats are illustrated. The incidence of adenocarcinoma was highest in the duodenoesophageal reflux group killed at
50 weeks (DER50 ¼ 60%).30 The earlier bile diversion was performed the fewer cancers developed (BD20 ¼ 3%; BD30 ¼ 19%),
suggesting that performing an antireflux procedure early may reduce the risk of subsequent esophageal adenocarcinoma.

operation, the animals were randomized into 3 treatment groups: (1)
surgical conversion of the esophagojejunostomy to a Roux-en-Y
esophagojejunostomy, which diverted the admixture of acid and bile
away from the esophagus, (2) acid-suppression therapy with PPI, or
(3) no further intervention. After 16 weeks of treatment, the rats were
killed and the distal esophagus was examined microscopically by an
experienced pathologist and scored for columnar mucosa length,
acute inflammation, chronic inflammation, columnar metaplasia,
and a composite Barrett’s metaplasia score calculated by adding
all the scores excluding the score for chronic inflammation. After
esophagojejunostomy, all rats developed columnar epithelium. Rats
treated with Roux-en-Y diversion had a statistically reduced length
of columnar mucosa, a decreased score of acute inflammation, a
decreased score of columnar metaplasia, and a decreased composite
Barrett’s metaplasia score in comparison to rats that received PPI
therapy or no therapy (Table 4). All of the 9 specimens from the
Roux-en-Y diversion group did not show an active inflammatory
infiltrate. There was widespread acute and chronic inflammatory
infiltrate in all 8 specimens from the no-therapy group and in 5 of the
8 specimens from the PPI therapy group. In all rats the columnar
epithelium near the esophagojejunal junction had scattered mucin-
secreting goblet cells compatible with Barrett’s esophagus. There
was no dysplasia or carcinoma noted in any specimen. This was
likely due to the short duration of duodenogastroesophageal reflux of
28 weeks in comparison to the more common duration of 40 weeks.
Rats treated with Roux-en-Y diversion had a lower esophageal pH
and bile acid concentration in comparison with preoperative values.
Those treated with PPIs had a higher esophageal pH and bile acid
concentration when compared with preoperative values. The authors
concluded that Roux-en-Y biliary diversion had the advantage of no
active acute or chronic inflammation and less bile acid exposure over
medical therapy.
Miyashita and colleagues32 reported a unique study on the
PPI, rabeprazole (Aciphex), which had shown promise to be a
chemopreventive agent against esophageal adenocarcinoma when
studied using the Levrat rat reflux model of esophageal

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Annals of Surgery  Volume 262, Number 6, December 2015 Animal Models of Esophageal Cancer

adenocarcinoma. They performed an esophagojejunostomy (Fig. 1)
on 40 rats, of which 17 were treated with a weekly subcutaneous
injection of rabeprazole starting 4 weeks postsurgery. The remaining
23 served as controls and were similarly injected with saline.32 After
40 weeks of treatment, only 29% of those treated with rabeprazole
developed esophageal adenocarcinoma compared with 74% of con-
trols (P < 0.05). In addition, Barrett’s esophagus developed in 100%
of the control group compared with only 65% of the rats treated with
rabeprazole. The authors concluded that rabeprazole protects against
Barrett’s esophagus and esophageal cancer and warrants further
investigation as a chemopreventive agent. They hypothesize that
the effect may be due to the known ability of rabeprazole to enhance
the viscosity of the esophageal mucin, thereby providing a protective
barrier against reflux-induced cellular damage.33
Effect of Chronic Inflammation in Carcinogenesis of
Esophageal Adenocarcinoma
An interesting observation by Nasr and colleagues29 reported
in a previous section of this report was that ‘‘PPI’s eliminated acute
esophageal inflammation, but unexpectedly had no effect on chronic
esophageal inflammation.’’ This would indicate that PPIs can ameli-
orate the acute inflammatory changes along with the acute symptoms
of GERD, whereas the chronic and therefore more insidious inflam-
matory changes persist. It is the chronic inflammation that is thought
to be the main stimulus or risk factor for the development of
cancers.34 Several investigators have shown that chronically sus-
tained activation of a chronic inflammatory response can produce the
microenvironment needed to generate the changes necessary for
metaplastic or carcinogenic cell transformation.35
Cyclooxygenase inhibitors, COX-1 and COX-2, have been
identified as key factors in the carcinogenic sequence of chronic
inflammation—Barrett’s esophagus—esophageal adenocarcinoma.
For this reason, COX-1 and COX-2 have become potential thera-
peutic targets and are the subject of considerable investigation.36– 39
Several studies have investigated COX-2 inhibitors as COX-2 is well
recognized to be overexpressed in Barrett’s esophagus. Oyama and
colleagues37 evaluated a group of 90 rats that underwent an esoph-
agojejunostomy as in the Levrat rat reflux model (Fig. 1). After the
operation, the rats were divided into a control group and an exper-
imental group fed celecoxib, a COX-2 inhibitor. The rats were killed
on the 10th, 20th, 30th, and 40th weeks of therapy, and their esophagi
were examined for esophagitis, columnar-lined epithelium, and
adenocarcinoma. In rats treated with celecoxib, adenocarcinoma
never developed and the incidence of esophagitis and columnar-
lined esophagus at each time interval was significantly lower than in
controls (P < 0.001).
The critical role of COX-2 in the progression of chronic
inflammation to adenocarcinoma was investigated by Buttar and
colleagues38 who carried out experiments similar to Oyama and
colleagues. They used the Levrat model to evaluate rats treated with
selective COX-2 and nonselective COX-1/COX-2 inhibitors. They
found that both selective and nonselective inhibitors decreased the
presence of inflammation, the development of Barrett’s esophagus,
and the incidence of esophageal adenocarcinoma compared with the
nontreated control rats.
Esquivias and colleagues39 compared the effect of indome-
thacin, a COX-1 and a COX-2 inhibitor, against MF-tricyclic, a
selective COX-2 inhibitor. A group of 112 rats underwent an
esophagojejunostomy as in the Levrat rat reflux model (Fig. 1) to
induce duodenogastroesophageal reflux. After recovery the rats were
randomized into 3 groups: no intervention, indomethacin therapy,
and MF-tricyclic therapy. Indomethacin treatment prevented the
development of intestinal metaplasia and esophageal adenocarci-
noma, whereas MF-tricyclic did not provide this protection (Fig. 11).
The authors concluded that indomethacin therapy decreases the
development of intestinal metaplasia and adenocarcinoma in the
rat reflux model. The general conclusion from these studies is that
efforts made to control the chronic inflammation associated with
gastroesophageal reflux are helpful in preventing the progression of
GERD to its complications of Barrett’s esophagus and esophageal
adenocarcinoma.
Effect of Oxidative Stress in the Carcinogenesis of
Esophageal Adenocarcinoma
The importance of oxidative stress induced by gastroesopha-
geal reflux as a mechanism of carcinogenesis in esophageal adeno-
carcinoma has been the subject of considerable study. The induction
of duodenogastroesophageal reflux using a modified rat reflux model
has been shown to produce reactive oxygen species and reactive
nitrogen species. These reactive species contribute to inflammation,
cause oxidative damage to DNA, proteins, and membrane lipids, and
are believed to contribute to carcinogenesis in the esophagus. In
theory, antioxidants should be able to inhibit oxidative stress and
function as potential cancer chemopreventive agents.40
Hao and colleagues41 investigated the antitumor effects of 2
antioxidants, a-tocopherol and N-acetylcysteine. Duodenogastroe-
sophageal reflux was induced with a modified rat reflux model.
The procedure consisted of making two 1.5-cm incisions, one
on the gastroesophageal junction and one on the antimesenteric
border of the duodenum, and then anastomosing the 2 incisions
together with mucosa to mucosa opposition. Two weeks after recov-
ery from the surgery, the rats were randomized into 3 groups: (1) An

TABLE 4. Histologic Comparison of Surgical (Roux-en-Y Bile Diversion) and Medical (PPI) Treatment of Duodenogastroesopha-
geal Reflux
Histologic Score REY (n ¼ 9) PPI (n ¼ 8) No Rx (n ¼ 8)
Columnar mucosal length, cm 0.44  0.06 0.92  0.8 1.17  0.31y
Acute inflammation score 0 3.67  0.33 1.67  0.21z
Columnar metaplasia score 0 3  0.45 2.33  0.76§
Composite Barrett’s score 20 10.67  0.67 8.83  1.42z
A vagal-preserving rat reflux model (esophagojejunostomy) was performed in all 30 rats to induce duodenogastroesophageal reflux. Three months after surgery, they were
randomized to 3 treatment groups to compare the effect of medical treatment with PPI acid suppression with surgical treatment with Roux-en-Yesophagojejunostomy. The control group
had no further intervention (no Rx). After 4 months, rats treated with REY diversion had a reduced length of columnar mucosa and decreased degrees of acute inflammation, columnar
metaplasia, and Barrett’s metaplasia (P < 0.03 to <0.001; see Table 4).

When REY and PPI compared, P < 0.001.
yWhen REY and no Rx compared, P < 0.03.
zWhen REY and no Rx compared, P < 0.001.
§When REY and no Rx compared, P < 0.01.
REY indicates Roux-en-Y; Rx, treatment.

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Greene et al Annals of Surgery  Volume 262, Number 6, December 2015

FIGURE 11. Comparison of selective and nonselective COX-1 and COX-2 inhibitors in preventing duodenogastroesophageal-induced
esophageal adenocarcinoma. Indomethacin, a nonselective COX-1/COX-2 inhibitor, prevents esophageal adenocarcinoma.39

a-tocopherol-treated group. This group consisted of several sub- underwent an esophagojejunostomy to induce duodenogastroeso-
groups treated with increasing doses of the drug. The subgroup phageal reflux or a control group that underwent an esophagojeju-
receiving the lowest dose served as the control group for the treated nostomy combined with a Roux-en-Y duodenal diversion to divert
rats. (2) An N-acetylcysteine-treated group. (3) A PPI (omeprazole)- bile away from the esophagus and allow only acidic gastroesopha-
treated group. A separate nonoperative control group was also geal reflux (Fig. 13). Half of the experimental groups were fed a
monitored. The rats were killed at 40 weeks, and their esophagi special diet containing 0.5% thioproline. The authors found that
were harvested for histopathologic examination. Before killing, the esophageal adenocarcinoma developed in rats with duodenogastroe-
gastric and duodenal contents from rats in the nonoperative control sophageal reflux and not in those with acidic gastroesophageal
group and the refluxate from rats with duodenogastroesophageal reflux. Oral administration of thioproline was able to inhibit the
reflux treated with an antioxidant or PPIs were collected, centrifuged, progression of Barrett’s esophagus to esophageal adenocarcinoma
and the pH of the supernatants measured. The investigators found after 45 weeks of exposure to duodenogastroesophageal reflux
that a-tocopherol reduced the incidence of esophageal adenocarci- (Fig. 13). A sequential comparative histologic study between the
noma in a dose-dependent manner to 59% in comparison with an
89% incidence in the low-dose control group (P ¼ 0.04). N-acetyl-
cysteine did not decrease rates of esophageal adenocarcinoma on its
own, but when combined with a-tocopherol, reduced the incidence to
55% (P ¼ 0.02). The surgically induced reflux was more acidic than
the duodenal contents and more alkaline than the gastric contents of
the nonoperated controls. In rats with surgically induced duodeno-
gastroesophageal reflux, the pH of reflux was higher in those treated
with omeprazole therapy than in those not treated. Even though
omeprazole was effective in producing acid-suppression, it had only
a weak nonstatistical tumor inhibitory effect. The investigators
concluded that (1) the findings of their study supported the
oxidative stress hypothesis of esophageal adenocarcinogenesis,
(2) antioxidants can reduce tumorigenesis and are potential agents
for cancer prevention, and (3) omeprazole has minimal, if any, tumor
inhibitory effect.

Effect of Endogenous Nitrates in Carcinogenesis of

Esophageal Adenocarcinoma
It has been hypothesized that endogenous nitrates found in the
gastrointestinal tract could become the carcinogens, causing esoph-
ageal adenocarcinoma. If true, thioproline, an effective nitrite-trap-
ping agent, may be beneficial in arresting the progression of Barrett’s
esophagus to adenocarcinoma (Fig. 12). Sasaki and colleagues42
studied 200 rats that were randomized to an experimental group and
FIGURE 12. Pathway illustrating endogenous nitrosation and
its prevention with thioproline, an effective nitrite-trapping
agent. Thioproline is nitrosated to nitrosothioproline, which
prevents the formation of carcinogenic N-nitroso compounds
by trapping nitrates. N-nitroso thioproline, which is nonmuta-
genic, is then excreted in the urine.42

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Annals of Surgery  Volume 262, Number 6, December 2015 Animal Models of Esophageal Cancer

FIGURE 13. Effect of thioproline on the

development of esophageal adenocarci-
noma in rats with duodenogastroeso-
phageal reflux. Thioproline (TPRO) is
an effective nitrite-trapping agent and
blocks endogenous nitrosation. Rats fed
thioproline during 45 weeks of esopha-
geal exposure to duodenogastroesopha-
geal reflux had fewer adenocarcinomas
than the control group (P < 0.01y).42

rats that received the thioproline diet and those that did not disclosed
a 3-step process to carcinogenesis: first, esophagitis, second, col-
umnar-lined esophagus, and third, adenocarcinoma. The investigator
concluded from their work: (1) the drug thioproline has an inhibitory
effect on duodenogastroesophageal reflux-induced adenocarcinoma
in rats; (2) these studies suggest that carcinogenesis could likely be
due to an endogenous duodenal nitrosation; and (3) carcinogenesis of
esophageal adenocarcinoma occurs in an inflammatory-metaplasia-
adenocarcinoma sequence.
Human Correlate
Studies with the rat reflux model have shown that the reflux of
an admixture of acid and bile results in the following: (1) It induces
Barrett’s esophagus with a similar histology and gene expression to
human Barrett’s mucosa. (2) It results in a chronic inflammation of
the Barrett’s mucosa that is not prevented or abated by PPI therapy.
(3) It creates tumors that are capable of continuous in vitro and in
vivo propagation, validating their neoplastic nature. (4) It induces a
mutational spectrum in the p53 tumor-suppressor gene similar to the
p53 mutations found in human gastrointestinal adenocarcinomas. (5)
It produces adenocarcinoma of the esophagus by the upregulation of
numerous genes known to be important in human esophageal adeno-
carcinoma. These findings strongly imply and studies in humans
strongly concur that the reflux into the esophagus of an admixture of
acid and bile is an important component in the pathogenesis of
human Barrett’s esophagus and the genesis of subsequent esophageal
adenocarcinoma.
Oberg and colleagues43 have shown in GERD patients that
repeated exposure to acidic gastric juice induces injury of the
squamous epithelium at the gastroesophageal junction that heals
with chronic-inflamed metaplastic cardiac epithelium. The presence
of cardiac epithelium is associated with deterioration of the lower
esophageal sphincter and encourages more reflux and a gradual
growth in the length of cardiac epithelium. Over time, some
patients develop intestinal metaplasia within the inflamed cardiac
epithelium. When compared with those who do not, both have
similar esophageal acid exposure but those with intestinal metaplasia
have a significantly higher bile exposure and a longer history of
GERD.44
Campos and colleagues45 performed a multivariate analysis of
502 patients with GERD, confirmed by pH monitoring, to identify
the risk factors for Barrett’s esophagus. The cohort consisted of 328
patients without Barrett’s esophagus, 67 with short-segment Barrett’s
esophagus (3 cm), and 107 patients with long-segment Barrett’s
esophagus (>3 cm).45 The investigators reviewed clinical, endo-
scopic, and physiologic data and found 7 factors that predicted
Barrett’s esophagus (Table 5). Multivariate analysis showed
increased esophageal bile exposure on the basis of monitoring
esophageal bilirubin was the most predominant independent pre-
dictive factor.
Stein and colleagues46 studied the relationship between gas-
troesophageal reflux, intestinal metaplasia, and adenocarcinoma in
the esophagus. These investigators measured esophageal acid
exposure on the basis of pH monitoring and bile exposure on the
basis of bilirubin monitoring in groups of patients with (1) reflux
esophagitis, (2) intestinal metaplasia of the esophagogastric junction,
(3) short-segment Barrett’s esophagus, (4) long-segment Barrett’s
esophagus, and (5) early esophageal adenocarcinoma.46 They
showed that long-segment Barrett’s esophagus and early esophageal
adenocarcinoma are clearly related to chronic increased exposure of
the distal esophagus to an admixture of acid and bile. To a lesser
degree, this seems also to be true for shorter segments of intestinal
metaplasia.
Nehra and colleagues47 measured bile acids in gastroesopha-
geal reflux by using a new technique of real-time high-performance
liquid chromatography combined with esophageal aspiration and pH
monitoring. They evaluated 10 asymptomatic and 30 symptomatic
GERD patients. Of the symptomatic patients, 10 had mucosal injury,
TABLE 5. Factors Associated With Barrett’s Esophagus
Variable Odds Ratio P
Bile reflux 4.3 0.0004
Hiatal hernia >4 cm 4.0 0.0001
Defective LES 2.6 0.0052
Number of reflux episodes >5 min 2.4 0.0006
Defective peristalsis 2.3 0.0004
Male sex 2.3 0.0011
Duration of symptoms >12 mos 2.0 0.0016
LES indicates lower esophageal sphincter.

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Greene et al
10 had erosive esophagitis, and 10 had Barrett’s esophagus. They
were able to detect cholic, taurocholic, and glycocholic bile acids
predominantly over a wide pH range. In those with erosive esoph-
agitis and Barrett’s esophagus, deoxycholic and taurodeoxycholic
bile acids predominated. The authors concluded that the pH of the
refluxed gastric juice modulates the toxic effects of bile acids by
influencing their solubility, and degree of ionization. This makes it
possible for esophageal mucosal damage to occur at a variety of
different pHs, with the most extensive injury because of the syner-
gism between the taurine-conjugated bile acid and the gastric acid.
Armstrong et al48 showed that at neutral pH all human bile
acids, except taurine-conjugated bile acids, induce mucosal damage.
They confirmed that acidification of the gastric juice led to precipi-
tation of unconjugated and glycine-conjugated bile acids and was
associated with diminished mucosal damage. The toxicity of taurine
conjugates was unaffected by acidification of the gastric juice.
Consequently, one patient may reflux bile that is more apt to induce
the genetic changes of Barrett’s mucosa in an acidic environment,
whereas another may reflux bile that is more apt to induce genetic
changes of the Barrett’s mucosa in a neutral pH environment. In
the former situation, acid-suppression therapy may be of benefit,
whereas in the latter situation, acid-suppression therapy may be
harmful. These findings in human patients are in agreement with the
findings of the rat reflux model.
Sikkema and colleagues49 performed a meta-analysis of
50 studies and showed that the baseline incidence of esophageal
adenocarcinoma under PPI therapy is 6 per 1000 patient-year follow-
up. The authors concluded that patients with Barrett’s esophagus, on
PPI therapy, are at low risk of malignant progression. Studies to prove
a potential cancer protective effect of PPIs on Barrett’s esophagus are
difficult because of the lack of an untreated control group. Nguyen
and colleagues50 evaluated 11,823 veterans with first-time diagnoses
of Barrett’s esophagus and a minimum follow-up of 2 years. They
found no significant difference between patients who developed
cancer and those who did not in the proportions of patients in each
group with filled PPI prescriptions. The analysis remained non-
significant when adjusted for concomitant use of nonsteroidal
anti-inflammatory drugs, aspirin, and statins. The additional use
of nonsteroidal anti-inflammatory drugs, aspirin, and statins con-
founded the results and prevented the authors from showing a
statistical benefit of PPIs in reducing the risk of esophageal adeno-
carcinoma in patients with Barrett’s esophagus.
A study by Hvid-Jensen and colleagues,51 performed in a
similar manner to the study of Nguyen, evaluated the cancer pre-
ventative effect of PPIs in 9883 patients with a new diagnosis of
Barrett’s esophagus followed up for more than 5 years. Their analysis
showed no cancer protective effects from PPIs. Rather, persistent and
long-term use of PPI was associated with a significantly increased
risk of adenocarcinoma or high-grade dysplasia. The authors con-
clude that until future studies can elucidate the association of long-
term PPI use with an increased risk of adenocarcinoma and high-
grade dysplasia, PPI therapy should be directed at symptom control
and other modalities should be considered for the prevention of
dysplasia and neoplasia. This is in line with national guidelines,
which recommend PPIs for symptom control and not the prevention
of esophageal adenocarcinoma.52,53
Chang and colleagues54 evaluated whether the incidence of
esophageal adenocarcinoma differs in patients with Barrett’s esoph-
agus who undergo antireflux surgery or medical treatment. The study
was a meta-analysis consisting of 700 surgical and 996 medically
treated patients followed up for 2939 and 3711 patient-years. The
incidence rate of esophageal adenocarcinoma was 2.8 per 1000
patient-years among surgically treated patients and 6.3 among
medically treated patients (P ¼ 0.034). When only controlled studies
922 | www.annalsofsurgery.com
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Annals of Surgery  Volume 262, Number 6, December 2015
were analyzed, the incidence rates were 4.8 and 6.5 per 1000 patient-
years in the surgical and medical patients, respectively (P ¼ 0.320).
The author concluded that the evidence suggesting that surgery
reduces the incidence of adenocarcinoma in patients with Barrett’s
esophagus is largely driven by uncontrolled studies.
Taken together, these human studies along with the rat reflux
model studied by Nishijima and colleagues30 indicate that once
Barrett’s esophagus develops, neither PPI therapy nor surgical anti-
reflux therapy can consistently eliminate the risk of esophageal
adenocarcinoma. To eliminate the risk of esophageal adenocarci-
noma, it seems that the pathway of chronic inflammation-metaplasia-
dysplasia needs to be interrupted before the formation of Barrett’s
metaplasia.
In summary, the studies in humans and rats clearly emphasize
that there is more to reflux than just acid. The reflux of an admixture
of gastric and duodenal juice over a wide extracellular range of pH
can be toxic. On entering the neutral pH intracellular environment,
bile acids can cause inflammation, stimulate cell proliferation,
induce CDx2 and NFkB expression, and have a mutagenic
effect—all important in the pathogenesis of Barrett’s esophagus
and esophageal adenocarcinoma.51,55 –57
Together, these studies indicate that long-term PPI therapy
should be discouraged, particularly in patients with Barrett’s esoph-
agus and a bilious component to their refluxed gastric juice.
Future Work and the Mouse Model of Esophageal
Adenocarcinoma
The rat reflux model has been the predominant animal model
to study GERD and esophageal adenocarcinoma over the past 35
years. Although it allows for good insight into the histology and
pathogenesis of esophageal adenocarcinoma, it is of limited value in
the study of specific genes. The available animal for this effort is the
transgenic mouse. Pham et al58 and Raggi et al59 have independently
shown that esophagojejunostomy is feasible in the mouse. Further-
more, Pham and colleagues58 have demonstrated that reflux in mice
leads to esophagitis and intestinal metaplasia, which has phenotypic
and molecular features of human Barrett’s esophagus. Xu and
colleagues60 have shown that the mouse model is able to generate
Barrett’s esophagus and esophageal adenocarcinoma at a comparable
rate to the rat reflux model. In addition, Fein et al61 demonstrated that
the gene TP53, which is frequently altered in human esophageal
adenocarcinoma, can be blocked in mice with duodenogastroeso-
phageal reflux to produce esophageal adenocarcinoma. The work of
Quante et al62 through a series of elegant experiments showed that
overexpression of IL-1b in a transgenic mouse exposed to bile acids
accelerates the development of Barrett’s metaplasia, dysplasia, and
neoplasia, with 40% of mice developing macroscopically visible
tumors. Electron microscopy of mouse Barrett’s metaplasia was
similar to that of the human. Both had nearly identical ultrastructure
features of columnar cell type, microvilli, granules, and mucins. PCR
studies showed that unconjugated bile acids induced gene promoter
demethylation, leading to upregulation of IL-6, Cdx2, and Notch1.
Subsequent histopathologic examination showed upregulation of
Cdx2, Notch1, and IL-6 in addition to TFF2 and Bmp4, all of which
are frequently upregulated in human Barrett’s esophagus and adeno-
carcinoma. Furthermore, gastric cardia progenitor cells seem to be
activated by bile acid IL-6-dependent inflammation, inducing
migration into the distal esophagus where they give rise to colum-
nar-like metaplasia and dysplasia. Together, these findings support
the concept that bile acids play a significant role in the pathogenesis
of Barrett’s esophagus and esophageal adenocarcinoma.62 Going
forward, the mouse reflux model, using transgenic mice, will play
a critical role in understanding the genetic basis of esophageal
adenocarcinoma.58,59,63,64
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Annals of Surgery  Volume 262, Number 6, December 2015
CONCLUSIONS
The rat reflux model, an esophagojejunostomy or its modifi-
cation to an esophagoduodenostomy, is a validated reproducible
model for the development of esophageal adenocarcinoma from
esophageal exposure to duodenogastric reflux. Reflux of an admix-
ture of duodenal and gastric juice into the rat’s esophagus results in
cellular and genetic changes that lead to the development of Barrett’s
esophagus and esophageal adenocarcinoma. Acid suppression in
such rats by surgical gastrectomy or medical therapy with PPIs
seems to potentiate the development of esophageal adenocarcinoma.
Anti-inflammatories, antioxidants, and nitrate-trapping agents
reduce the incidence of adenocarcinoma in the rat reflux model.
Antireflux surgery performed in rats with Barrett’s esophagus does
not eliminate the risk of adenocarcinoma but does so when performed
before the development of Barrett’s esophagus.
Even though studies using the rat reflux model have shown,
over and over again, that duodenogastroesophageal reflux containing
bile acids is associated with the development of esophageal adeno-
carcinoma, they do not prove that the same association occurs in
humans. Definitive proof of the same association in humans would
require a large-scale randomized control trial that would knowingly
put a group of human subjects at risk. As this violates all non-
maleficent principles of medicine, it is an impossible association to
prove. If one accepts that the laws governing the biology of the rat are
fundamentally the same as those governing the biology of the human,
the insights provided by the rat reflux model enable a better under-
standing of human GERD and its progression to esophageal adeno-
carcinoma.
In humans, it has not been unequivocally established whether
PPIs promote or prevent the development of esophageal adenocar-
cinoma in patients with Barrett’s esophagus or that antireflux surgery
reduces the incidence of adenocarcinoma in patients with Barrett’s
esophagus. An important, largely unanswered question is whether
antireflux surgery performed before the development of Barrett’s
esophagus prevents the development esophageal adenocarcinoma as
suggested by the rat reflux model. A singular study carried out to
assess this concept showed that Barrett’s esophagus was prevented
with surgery but not, as would be expected, with PPIs.65 What we do
know unequivocally is that rats and humans who do not reflux rarely,
if ever, develop Barrett’s esophagus and subsequent adenocarcinoma.
Therefore, on the basis of studies from the rat reflux model and
studies on human patients, we propose that to prevent esophageal
adenocarcinoma in humans, the reflux of bile and acid must be
controlled before the development of Barrett’s esophagus by methods
other than chronic PPI therapy.
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