GASTROENTEROLOGY 1998;115:642–648
Helicobacter pylori Infection Induces Gastric Cancer
in Mongolian Gerbils
TAKESHI WATANABE,* MAYUMI TADA,‡ HIROFUMI NAGAI,*
SATOSHI SASAKI,* and MASAFUMI NAKAO‡
*Drug Safety Research Laboratories and ‡Pharmacology Laboratories, Takeda Chemical Industries, Ltd., Osaka, Japan
See editorial on page 780.
Background & Aims: Although epidemiological studies
have indicated that Helicobacter pylori infection plays
a crucial role in gastric carcinogenesis in humans,
there is no direct proof that H. pylori is actually
associated with gastric carcinogenesis. The purpose of
this study was to elucidate the relationship between
H. pylori infection and gastric carcinogenesis using an
animal model of long-term H. pylori infection. Methods:
Mongolian gerbils were orally inoculated with H. pylori,
and the sequential morphological changes in the stom-
ach were examined for up to 62 weeks. Results:
H. pylori was constantly detected in all infected ani-
mals throughout the study. At the 26th week, severe
active chronic gastritis, ulcers, and intestinal metapla-
sia could be observed in infected animals. By the end
of the study, adenocarcinoma had developed in the
pyloric region of 37% of the infected animals. All
tumors consisted of well-differentiated intestinal-type
epithelium, and their development seemed to be closely
related to intestinal metaplasia. Conclusions: We have
successfully demonstrated that long-term infection
with H. pylori induces adenocarcinoma in Mongolian
gerbils. The observations are thus highly suggestive of
the involvement of H. pylori infection in gastric carcino-
genesis in humans.
lthough the overall incidence of gastric cancer has
A steadily declined over the past 50 years, it is still a
major health problem and remains the second most
common cancer in the world.1 Since the discovery of
Helicobacter pylori in 1983,2 this microorganism has been
implicated in gastric carcinogenesis on the basis of
various epidemiological studies.3–7 A Working Group of
the World Health Organization International Agency for
Research on Cancer concluded in 1994 that H. pylori is a
group 1 carcinogen in humans.8 However, the prevalence
of H. pylori in patients with gastric carcinoma still
remains considerably variable among the studies,9 and
there has been no direct demonstration that H. pylori is
actually associated with gastric carcinogenesis. Gastric
cancer is a multifactorial disease10; therefore, genetic
factors and environmental factors other than H. pylori can
complicate the elucidation of the association between
H. pylori infection and gastric cancer. We believe that
studies using animal models of H. pylori infection,
especially those following the effects of long-term infec-
tion on the gastric mucosa, should be able to shed further
light on this unresolved issue. The Mongolian gerbil
(Meriones unguiculatus) is an appropriate animal in which
various gastrointestinal diseases such as gastritis and
ulcers that mimic human H. pylori infection can be
studied.11,12 In our current research, we therefore exam-
ined the pathological consequences of long-term H. pylori
infection using specific pathogen–free Mongolian gerbils
with the aim of clarifying the association of H. pylori and
gastric carcinogenesis.
Materials and Methods
Five-week-old male specific pathogen–free Mongolian
gerbils (MGS/Sea) were purchased from Seiwa Experimental
Animals (Fukuoka, Japan) and maintained under standard
laboratory conditions (room temperature, 23°C 6 2°C; rela-
tive humidity, 55% 6 5%; 12/12-hour light/dark cycle) with
free access to a commercial rodent diet (CE-2; Clea Japan,
Tokyo, Japan) and tap water. H. pylori TN2GF4 used in this
study had been isolated from a patient with gastric ulcer and
was motile and urease, catalase, and oxidase positive. The strain
produced vacuolating cytotoxin and contained the cytotoxin-
associated gene (cagA). H. pylori for the experimental inocula-
tion was grown in brucella broth supplemented with 2.5%
heat-inactivated fetal bovine serum in GasPak jars (BBL,
Cockeysville, MD) containing CampyPak (BBL) at 37°C for
about 24 hours. Sterilized glycerol was added to the cultures at
a final concentration of 15%, and cultures were kept at 280°C
until use. Fifty-five animals were inoculated with 1 mL of
broth culture containing 107.54 colony-forming units (CFU) of
H. pylori by intragastric gavage after fasting for 24 hours.
Abbreviation used in this paper: AB-HID, alcian blue with high-iron
diamine.
r 1998 by the American Gastroenterological Association
0016-5085/98/$3.00
September 1998 HELICOBACTER PYLORI INDUCES GASTRIC CANCER 643

Thirty animals served as uninfected controls. Eight of the
infected animals died between 40 weeks after inoculation and
the end of the experiment. Data from these animals were
excluded from analysis because of autolytic changes in the
stomach. Six, 26, 39, and 52 weeks after inoculation, 5 infected
animals were killed, and, in addition, all surviving animals, 27
infected and 30 uninfected animals, were killed 62 weeks after
inoculation. At each time point, one half of the stomach from 5
infected animals was used for the quantitative determination of
the bacterium. The other half of the stomach from these 5
animals and the entire stomach from the other animals, killed
62 weeks after inoculation, were examined for sequential
morphological changes. After the stomach was opened along
the greater curvature, the longitudinal half of the stomach was
homogenized with physiological saline. An aliquot of dilutions
was inoculated onto modified Skirrow’s agar and incubated at
37°C for 4 days under microaerobic conditions. The density of
infection was estimated by counting the number of colonies per
plate and expressed as log CFU per gastric wall. For histologi-
cal examination, the stomach was stapled onto paper and fixed
in 10% neutral buffered formalin. Fixed tissue was cut into
longitudinal strips, about 3 mm wide, for histopathologic
examination of the entire gastric mucosa. After processing for
histology by routine methods, paraffin-embedded sections were
cut and stained with H&E; for mucus clarification or for
demonstration of neuroendocrine cells, when necessary, alcian
blue (pH 2.5) with high-iron diamine (AB-HID) stain or
Grimelius’ and Sevier–Mungar stains were applied. Histopatho-
logic lesions of the glandular stomach were categorized as
follows: (1) active chronic gastritis (characterized by severe
inflammatory cell infiltration and multiple lymphoid follicle
formation throughout the pyloric region and part of the fundic
region); (2) ulcer; (3) regenerative hyperplasia; (4) invagination
of glands into the submucosa; (5) hyperplastic polyp; (6)
intestinal metaplasia (diagnosed by the presence of goblet cells
that were positive for AB-HID staining); (7) adenocarcinoma
(diagnosed by the presence of atypical glands that locally
invaded the muscle layer and destroyed the original architec-
ture); and (8) carcinoid (diagnosed by neuroendocrine nests in
excess of three glands in diameter). In addition, immunohisto-
chemical staining to detect expression of p53 protein was
performed. Formalin-fixed, paraffin-embedded sections from
animals diagnosed with adenocarcinoma and from 5 uninfected
animals killed 62 weeks after inoculation were pretreated by
autoclaving (105°C, 10 minutes) in a target unmasking fluid
(Monosan; Uden, The Netherlands). After routine inhibition of
endogeneous peroxidase activity with 0.3% H2O2 in methanol,
nonspecific proteins were blocked with normal goat serum.
Specimens were incubated with a monoclonal antibody reactive
to wild-type and mutant-type p53 protein (Dako Japan Corp.,
Kyoto, Japan). Immunolocalization was demonstrated using
the Envision method (Dako Japan Corp.) with diaminobenzi-
dine as the substrate. Only distinctly increased nuclear staining
was regarded as positive, and when staining was observed in all
or nearly all tumor cells, tumors were considered positive.
Negative control tissues were prepared in exactly the same way
as described above, except the primary antibody was omitted.
All experimental protocols described were conducted in
accordance with the Guidelines for the Care and Use of
Laboratory Animals in Pharmaceutical Research Division,
Takeda Chemical Industries, Ltd., and approved by the Ethical
Committee for the animal experiments of our division.
Results
H. pylori was detected in all infected animals
throughout the study for up to 62 weeks. The bacterial
count reached 105.17 CFU/gastric wall at 6 weeks and
then remained constant until the end of the experiment
(Table 1). Sequential histopathologic changes are summa-
rized in Table 1. By the 6th week, severe active chronic
gastritis, characterized by dense neutrophil and mono-
nuclear cell infiltration, and multiple lymphoid follicle
formation in both the mucosa and submucosa could be
observed in all infected animals (Figure 1). The gastritis

Table 1. Sequential Microbiological and Histopathologic Changes in the Glandular Stomach of Mongolian Gerbils
Infected With H. pylori
Duration after HP inoculation (wk) 6 26 39 52 62
No. of animals examined 5 5 5 5 27 a
Microbiology
Bacterial count (log CFU/gastric wall) 5.17 6 0.41b 5.33 6 0.98 5.89 6 0.51 4.59 6 1.45 5.40 6 0.95
Histopathology
Active chronic gastritis 5 c (100) 5 (100) 5 (100) 5 (100) 27 (100)
Ulcer 0 (0) 5 (100) 5 (100) 2 (40) 16 (59)
Regenerative hyperplasia 5 (100) 5 (100) 5 (100) 5 (100) 27 (100)
Invagination of glands 0 (0) 5 (100) 5 (100) 5 (100) 27 (100)
Hyperplastic polyp 0 (0) 0 (0) 0 (0) 3 (60) 4 (15)
Intestinal metaplasia 0 (0) 3 (60) 4 (80) 5 (100) 23 (85)
Adenocarcinoma 0 (0) 0 (0) 0 (0) 0 (0) 10 (37)
Carcinoid 0 (0) 0 (0) 0 (0) 0 (0) 3 (11)

HP, H. pylori.
a Microbiological examination was performed on 5 animals.
b Mean 6 SD.
c Incidence with percentage in parentheses.
644 WATANABE ET AL. GASTROENTEROLOGY Vol. 115, No. 3

Figure 1. (A ) Active chronic gastritis observed in the pyloric region of

Figure 2. (A) Intestinal metaplasia observed near the ulcer in Mongolian
Mongolian gerbils 6 weeks after H. pylori inoculation. (B ) The gastritis
gerbils 39 weeks after H. pylori inoculation. (B) The metaplastic glands are
is characterized by severe neutrophil and mononuclear cell infiltration
composed of columnar cells and a large number of goblet cells producing
in both the mucosa and submucosa. The normal mucosal architecture
sialomucins (blue) and occasionally sulfomucins (gray) (H&E [A] and
is almost completely lost and replaced by regenerative hyperplastic
AB-HID [B]; original magnification 653 [A] and 1303 [B]).
epithelium. The thickness of the mucosa increased remarkably
compared with that of an uninfected animal (B) (H&E; original
magnification 653). submucosal cystic glands. These metaplastic glands ap-
peared more atypical than the surrounding nonmetaplas-
originated in the pyloric antrum, extended to the fundic tic, hyperplastic glands (Figure 3). Budding of glands,
region, and was observed continuously throughout the enlarged and vesicular nuclei with prominent nucleoli,
study. The normal mucosal architecture was almost and stratification of nuclei were frequently observed. By
completely lost and was replaced by hyperplastic epithe- the end of the study, adenocarcinoma had developed in 10
lium. The thickness of the mucosa increased remarkably, of the remaining 27 (37%) infected animals. Grossly,
and pseudopyloric gland formation and invagination into almost all tumors were located in the pyloric region and
the submucosa of hyperplastic glands were noted at the detected as crater-like areas with irregular elevated
fundus-pylorus border. After the 26th week, ulcers, ridges. Gastric wall thickening was frequently observed
extending to the muscular layer, developed in the region in these regions. The histomorphological features of these
of the fundus-pylorus border of all infected animals. tumors were quite similar (Figure 4). The tumors arose in
Epithelial hyperplasia progressed, and intestinal metapla- the areas of intestinal metaplasia in deep mucosa or
sia was observed in the mucosa of the pyloric region, submucosa and grew downward. They penetrated into
especially in the area close to the ulcer. These metaplastic the muscle layer and occasionally spread into the serosa.
glands were composed of columnar cells containing little Metastasis or vascular invasion, however, was not ob-
or no mucus and a moderate number of goblet cells
producing sialomucins and occasionally sulfomucins (Fig-
ure 2). In addition, paneth cells were occasionally
observed in some of the metaplastic glands. Cystic
dilatation of the invaginated glands in the submucosa was
a common finding during this period. These cystic
submucosal glands were mainly lined by low, columnar,
mucous cells with little cellular atypia. From the 39th to
52nd week, the structural irregularity of the hyperplastic
glands was more prominent, and complex branching or
cystic dilatation of these glands was frequently observed.
Hyperplastic polyps, characterized by hyperplasia of the
tall columnar surface epithelium and prominent edema,
were also noted in the fundus. Despite such structural Figure 3. (A ) Intestinal metaplasia observed in the pyloric region of
irregularities, little cellular atypia could be observed in Mongolian gerbils 52 weeks after H. pylori inoculation. (B ) The
metaplastic glands show complex branching and budding, and en-
the hyperplastic epithelium. The frequency of intestinal
larged and vesicular nuclei with prominent nucleoli and stratification
metaplasia increased during this period and could be of nuclei are frequently recognized (H&E; original magnifications 503
observed not only in the mucosa but also in the [A ] and 1303 [B ]).
September 1998 HELICOBACTER PYLORI INDUCES GASTRIC CANCER 645

Figure 4. (A ) An adenocarcinoma observed in the pyloric region of a Mongolian gerbil 62 weeks after H. pylori inoculation. (B ) The tumor has
arisen in the area of intestinal metaplasia in the deep mucosa, and the direction of growth is downward. Penetration into the lamina muscularis
and destruction of the original architecture can be observed. (C ) Spreading into the serosa of the neoplastic glands is also noted, and these
glands show nuclear pleomorphism. (D ) The neoplastic glands are composed of intestinal-type epithelium, which contains goblet cells producing
predominantly sialomucins (blue). Cystic dilatation of the neoplastic glands with an accumulation of mucus and extracellular mucus pools are
frequently seen in the submucosa. (E ) Immunohistochemically, almost all tumor cells express p53 protein (H&E; original magnifications 303 [A ],
653 [B ], 1303 [C ]; AB-HID, 1303 [D ]; p53, 1303 [E ]).
646 WATANABE ET AL.
served in any case. The tumors retained a well preserved
glandular structure and consisted of well-differentiated
columnar intestinal-type epithelium, which contained a
moderate to large number of goblet cells producing
predominantly sialomucins. Irregular budding or branch-
ing of glands and nuclear pleomorphism were frequently
observed. Copious mucous production was a characteris-
tic feature, and cystic dilatation of neoplastic glands with
an accumulation of mucus and extracellular mucus pools
were commonly seen in the submucosa. The stroma of
these tumors showed various degrees of inflammation and
fibrosis, and osseous metaplastic foci were occasionally
noted. Immunohistochemically, intranuclear overexpres-
sion of p53 protein was observed in 4 of the 10 (40%)
adenocarcinomas. In these cases, p53 protein–expressing
cells were densely distributed in almost all tumor cells.
The relationship between p53 protein expression and
tumor histology, including the invasive pattern, was not
clear. There was no increase in immunoreactivity in the
uninfected animals, and negative controls stained as
expected. In addition to the adenocarcinoma, carcinoids
derived from fundic mucosa were also detected in 3
infected animals. Tumor cells were moderately pleomor-
phic, with pale or eosinophilic cytoplasm and argyro-
philic, and were arranged in solid sheets or small nests in
lamina propria. One of three carcinoids spread into the
submucosa. No other hyperplastic changes in neuroendo-
crine cells were observed in any infected animal. In the
uninfected animals, no significant changes had been
observed by the end of the study.
Discussion
We successfully showed that long-term infection
with H. pylori induces gastric adenocarcinoma in Mongo-
lian gerbils. The tumor incidence was 37% (10 of 27
animals) 62 weeks after H. pylori inoculation. Although
H. pylori infection has been categorized as a risk factor for
gastric cancer based on the results of various epidemiologi-
cal studies,3–7 no direct evidence of this relationship has
been presented to date. In animal studies, Fox et al.13
reported that Helicobacter mustelae, a relative of H. pylori, is
associated with gastric carcinogenesis in naturally in-
fected ferrets. To the best of our knowledge, our study is
the first showing a direct relationship between H. pylori
infection and gastric carcinogenesis.
All tumors induced in the H. pylori–infected Mongo-
lian gerbils showed similar histological features. They
were located in the pyloric region and consisted of
well-differentiated intestinal-type epithelium, correspond-
ing to intestinal-type adenocarcinoma in humans. In
comparing the histology of the cancer associated with
GASTROENTEROLOGY Vol. 115, No. 3
H. pylori infection in humans and Mongolian gerbils, the
topographic location was similar.6,14–17 The histological
subtype of cancer is difficult to assess accurately because
of the existing controversy about the difference between
intestinal and diffuse types of gastric cancer in their
association with H. pylori infection in humans.5–7,14–17
The reason for these conflicting observations is consid-
ered to be mainly the different methods used for the
detection of H. pylori.15 In studies based on histological
detection of H. pylori, the prevalence has generally been
higher in the intestinal rather than in the diffuse cancer
type.7,18,19 This difference has been less distinct or
nonexistent in most serological studies,5,6,14,17 although
there are some exceptions.15,16 However, in focusing on
early gastric cancer to eliminate any possible complicat-
ing effects of tumor progression on colonization with
H. pylori, the prevalence of H. pylori infection is signifi-
cantly higher in intestinal-type gastric cancer regardless
of the method used to detect H. pylori infection.16,20,21 In
addition, several reports have shown that H. pylori
infection and intestinal-type gastric carcinoma share
some similar epidemiological characteristics.15 The patho-
genesis of gastric cancer is certainly multifactorial, and
genetic host and/or environmental factors, other than H.
pylori, may complicate the resolution of this issue in
humans. It seems to be important, therefore, that tumors
induced in the H. pylori–infected Mongolian gerbils, in
which other environmental factors could be minimized,
were all intestinal-type adenocarcinoma.
Epidemiological surveys,22,23 histopathologic examina-
tion,24 and biochemical25 and genetic26 analyses have
shown that intestinal metaplasia, especially the sulfomu-
cin-secreting type, may be a possible precancerous lesion
leading to intestinal-type adenocarcinoma in humans.27
In the H. pylori–infected Mongolian gerbils, intestinal
metaplasia occurred in the mucosa or submucosal invagi-
nated glands in the pyloric region before the cancer
developed. Although these metaplastic glands produced
sialomucins predominantly, the metaplastic epithelium
was more dysplastic than the surrounding nonmetaplastic
hyperplastic epithelium, and the tumors arose in associa-
tion with metaplastic epithelium. Furthermore, all tu-
mors found in the present study consisted solely of
intestinal-type epithelial cells. All these results indicate a
close relationship between intestinal metaplasia and
gastric carcinogenesis, and it can be concluded that
intestinal metaplasia is a precancerous lesion leading to
gastric cancer in this model.
Intestinal metaplasia has been widely assumed to be
linked to chronic gastritis with mucosal atrophy.28
Although the pathogenesis of intestinal metaplasia has
not been clarified as yet, Scott et al.29 suggested that a
September 1998
hyperproliferative state in the inflamed gastric mucosa
may promote the progression from normal mucosa to
metaplastic epithelium. Moreover, Recavarren-Arce et
al.30 reported that an altered gastric microflora, subse-
quent to hypochlorhydria due to atrophy, increases
N-nitroso compounds in the gastric lumen, and these
substances may induce intestinal metaplasia.30 In our
study, so-called mucosal atrophy, characterized by a
decrease in mucosal height and gland density, was not
evident; however, the original glands, including oxyntic
glands, were almost completely lost and were replaced by
hyperplastic epithelium. This mucosal state would cause
the generation of metaplastic epithelium. Metaplastic
epithelium was frequently noted around the ulcers;
therefore, ulcer formation might have contributed to
intestinal metaplasia in this model, possibly by enhance-
ment of the mucosal proliferative activity in its regenera-
tive process.
No significant changes were noted in uninfected
animals in this study. Also, severe gastritis in
H. pylori–infected Mongolian gerbils can be reversed by
clearance of the organism.12 This indicates that H. pylori
infection alone caused severe inflammatory changes includ-
ing intestinal metaplasia, and may also induce gastric
cancer. Several animal models for H. pylori produce severe
gastritis, including gnotobiotic piglets, monkeys, and
mice.31–33 However, induction of ulceration or intestinal
metaplasia has never been reported in these models,
although higher levels of colonization were noted in some
of them.33 Possibly host factors such as differences in
mast cell subtype or cytokine production are responsible
for the susceptibility of Mongolian gerbils to H. pylori.12
Although the pathogenesis of the tumor is unknown,
there are several possible mechanisms by which H. pylori
infection may play a part in the mutagenesis of the gastric
epithelium. One explanation is increased epithelial turn-
over in the inflamed mucosa. Enhanced cell replication
increases the frequency of mutation by errors in gene
replication, by conversion of endogenous or exogenous
DNA adducts to mutation, or by diminishing the time
for the DNA repair processes.34 Cahill et al.35 have stated
that gastric mucosal cell proliferation is significantly
higher in patients with H. pylori–positive gastritis than in
those with H. pylori–negative gastritis. Recently, a novel
cell proliferation gene ( pag) has been shown to share
approximately 50% homology with a 26-kilodalton
antigen of H. pylori.36 In the Mongolian gerbil model,
prominent regenerative hyperplasia was a characteristic
feature and constantly observed throughout the study.
This was suggestive of persistently and highly enhanced
cellular proliferation in the gastric mucosa in this model.
Therefore, H. pylori itself and/or an indirect hyperplastic
HELICOBACTER PYLORI INDUCES GASTRIC CANCER 647
response to cellular damage or inflammatory products
stimulate the proliferation of gastric epithelial cells and
may cause an increased opportunity for mutation.37 An
additional explanation is the effect of the formation of
nitric oxide, a putative endogenous mutagen in the
presence of oxide. It has been reported that a soluble
protein from H. pylori enhances the expression of the NO
synthase gene38 and NO production in macrophage cell
lines.39 In addition, high concentrations of NO induce
wild-type p53 protein accumulation,40 and the NO-
related deamination of DNA has been reported to cause
GC-AT transitions, which are frequently found in p53
mutations in gastric cancer.39 The overexpression of p53
protein in gastric cancer observed in the present model,
may therefore suggest the participation of NO in the
gastric carcinogenesis in H. pylori–infected Mongolian
gerbils.
We have successfully shown that long-term H. pylori
infection induces gastric cancer in Mongolian gerbils. All
of the induced tumors were located in the pyloric region
and consisted of well-differentiated intestinal-type epithe-
lium. The development of these tumors seemed to be
closely related to intestinal metaplasia formed in the
chronically inflamed mucosa. Although careful interpre-
tation is required in extrapolating the data from such an
animal model to humans, our present findings are highly
suggestive of the involvement of H. pylori infection in
gastric carcinogenesis in humans.
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Received October 28, 1997. Accepted May 5, 1998.
Address requests for reprints to: Takeshi Watanabe, M.D., Drug
Safety Research Laboratories, Takeda Chemical Industries, Ltd.,
17-85, Jusohonmachi 2-chome, Yodogawa-ku, Osaka 532-8686,
Japan. Fax (81) 6-300-6918.
The authors thank Jeffrey A. Hogan, Leslie I. Brezak, Toshimasa
Kyutoku, Yasuharu Yamamoto, Naomi Inui, and Yuri Ozaki for their
helpful assistance.