[CANCER RESEARCH 58, 4255-4259. October I, 1998]

Advances in Brief

Development of Helicobacter pylori-induced Gastric Carcinoma inMongolian Gerbils1

Shoji Honda,2 Toshio Fujioka, Masashi Tokieda, Ryugo Satoh, Akira Nishizono, and Masaru Nasu

Second Department of Internal Medicine {S. H.. T. F.. M. T., R. S.. M. /V./ and Department of Microhiologv {A. N.], Otta Medical University, Hasama-machi, Otta 879-5593.

Japan

Abstract

Helicobacter pylori is classified by IARCAVHO as a definite humangastric carcinogen, despite "inadequate experimental evidence." To obtain

direct evidence concerning this relationship, we investigated the his-

topathological findings of gastric mucosa using a model of //. pyloriinfection in Mongolian gerbils. The animals were challenged p.o. with //.pylori ATCC-43504 and sacrificed at 6, 12, and 18 months after inocula

tion for histológica! examination. All inoculated animals were infectedwith //. pylori. Severe infiltration of the lamina propria by polymorpho-

nuclear and mononuclear cells appeared in the lesser curvature of theantrum, with an increase in epithelial cell proliferation, and the infiltration extended to the body. Atrophie gastritis and focal intestinal metaplasia also appeared in the lesser curvature of the animi mucosa at 6 monthsafter inoculation. Intestinal metaplasia became severe, with dysplasia,after that. At 18 months after II. pylori inoculation, two of five infectedanimals showed three well-differentiated gastric cancers. The uninfected

control animals showed no abnormal findings throughout the entire observation period. Here, it was confirmed that //. pylori infection alonecauses gastric cancer in an animal model.

Introduction

A relationship between Helicobacter pylori infection and gastriccancer of both intestinal and diffuse types has been suggested inserological studies (1, 2), and two pathways have been proposed toexplain the role of H. pylori infection and gastric cancer in both types(3). Evidence from an animal model is needed to prove this relationship. It has been reported that H. pylori infection causes atrophiegastritis and intestinal metaplasia in Mongolian gerbils (4, 5). Atrophyand intestinal metaplasia are considered precursors of intestinal-type

gastric cancer (6). We observed histological changes in the stomachsof Mongolian gerbils infected with H. pylori and compared the resultswith those from uninfected control animals to investigate the relationship between H. pylori infection and gastric cancer.

Materials and Methods

Animals and Infection Model. Five-week-old male Mongolian gerbilsweighing 30-40 g (Seac Yoshitomi Co. Ltd.. Fukuoka, Japan) were fasted for

24 h and then fed chow (Oriental Yeast Co., Tokyo, Japan) and water adlibitum beginning 12 h after H. pylori inoculation. Mongolian gerbils wererandomly divided into two groups and challenged p.o. with vehicle or IO9

colony-forming units of H. pylori ATCC-43504, expressing the cagA gene and

vacuolating cytotoxin, as described previously (5). Four or five animals in eachexperimental group were weighed and sacrificed under anesthesia with ether at6, 12, or 18 months after inoculation. Immediately after killing, the blood wasdrawn from the heart, and serum was obtained and stored at —¿�20°Cuntil

Received 6/30/98; accepted 8/19/98.The costs of publication of this article were defrayed in part by the payment of page

charges. This article must therefore be hereby marked advertisement in accordance with18 U.S.C. Section 1734 solely to indicate this fact.

' This work was supported in part by Grant-in-Aid for Science Research 98457170

from the Ministry of Education. Science and Culture (Japan).2 To whom requests for reprints should be addressed. Phone: 81-975-86-5804; Fax;

81-975-49-4245; E-mail: shonda@oita-med.ac.jp.

testing for ami-//, pylori antibody using ELISA method, as described previ

ously (5). The stomach was quickly removed and used for histological examinations.

Experiments were performed according to the guidelines of Ethical Committee for Animal Experiments at Oita Medical University (Oita. Japan).

Histological Examinations. The stomach was fixed in 10% neutral andisotonic buffered formalin for 4 h. The stomach was cut up into eight longitudinal parts and embedded in paraffin. The 5-jnm sections were stained withH&E, Giemsa, PAS.3 and Alcian blue (pH 2.5) stains and examined for

inflammatory response with or without intestinal metaplasia and the presenceof organisms.

Diagnosis of Dysplasia and Adenocarcinoma. Gastric dysplasia was diagnosed according to the following pathological criteria (7): increasing proliferation of the cells, abnormal morphology, and pleomorphism of the cells,architectural derangement of glands, and stromal changes. Gastric adenocar-

cinoma was diagnosed by the presence of atypical glands that invaded into theproper muscular layer.

Statistical Analysis. All values are expressed as the means ±SD. Datawere analyzed by the Student's / test. Significance was defined as P s 0.05.

Results

Normal Histology of the Glandular Stomach of MongolianGerbils. Normal gastric mucosa of a Mongolian gerbil is shown inFig. 1. All superficial epithelial cells, pylorie glands, and some cellsof the fundic glands were stained by PAS stain. Pyloric glands werealso stained by Alcian blue (pH 2.5). Superficial epithelial cells andfundic glands were not stained by Alcian blue stain. The uninfectedcontrol animals showed no abnormal findings throughout the entireobservation period.

Body Weight and IgG Anti-W. pylori Antibody Titer. All inoculated animals showed high titers of the serum anti-A/. pylori anti

body, as compared to uninfected control animals, throughout theentire observation period, and the body weights of these animals weresignificantly lower than those of control animals at 6 months afterinoculation (Table 1).

3 The abbreviations used are: PAS, periodic acid-Schiff; PMN. polymorphonuclear:

MN, mononuclear.

Table 1 Effect of H. pylori infection on hotly weight unti anti-H. pylori tmtihody liter

in Mongolian gerhils

GroupControlH.

p\lohInvestigation

time(months)6121861218Body

weight<g>"86

±5.486±11.4100±7.069±9.6'76

±13.482±19.2Antibody

titer(units)"2.12.23.918822890.90.60.4"89C2242''2707

889r-'

" Results represent mean ±SD for four or five animals.*P < 0.05 versus 6 and 12 months.' P < 0.01 versus control at the same time investigation time point.d P < 0.05 l'ir««control at the same time investigation time poini.' P < 0.01 versus 6 months.

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Fig. I. Microscopie views of the gastric mucosa of 6-month-old uninfected Mongolian gerbils. All epithelial cells and some fundic gland cells were PAS positive but not Alcianblue positive. Only pyloric glands were positively stained by both PAS and Alcian blue. A-C, body: D-F. antrum. A and D. H&E stain; original magnification. X25. B and £.PASslain; original magnification. X25. C and F. Alcian blue (pH 2.5); original magnification. X25.

Inflammatory Cell Infiltration and Gastric Ulcers. Microscopicexamination showed severe infiltration of the lamina propria by PMNand MN cells in the antral mucosa. Although PMN and MN cellinfiltration was first localized in the lesser curvature of antrum, itextended to the lesser curvature of body at 12 months after inoculation

and to the greater curvature at 18 months. Gastric ulcers were continuously observed from 6 to 18 months after H. pylori inoculation(Table 2). They localized in area of the pyloric glands, and almost allof them penetrated the submucosa or the proper muscular layer. Inaddition, some penetrated the pancreas or liver.

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Table 2 Hlstopathologícaíchanges of the gasine mucosa in stomachs of Mongoliangerhils infected with H. pylori ATCC-43504"

Helicohmler /ir/»ri-INDUCED GASTRIC CANCER

No. of positive cases/lotal no. of cases formice sacrificed at:

HistopathologicalfindingsGastritisGastric

ulcerAtrophiegastritisIntestinalmetaplasiaDysplasiaGasine

cancer6

months5/54/54/52/50/50/512months4/43/44/43/42/40/418months5/55/55/55/55/52/5"

1The uninfected control animals (n = 5 in each case) showed no abnormal Undings.' Three well-differentiated adenocarcinomas were observed in two of five animals.

Atrophy and Intestinal Metaplasia. The mucosa infiltrated withPMN and MN cells was hyperplastic first (Fig. 2). and the number ofproper gastric glands was reduced after that. Atrophie gastritis andfocal intestinal metaplasia appeared in the lesser curvature of antruniat 6 months after inoculation, and it extended to the body and greatercurvature following inflammation. Intestinal metaplasia became severe and was associated with dysplasia at 12 and 18 months after H.pylori inoculation (Fig. 3).

Occurrence of Gastric Cancer. At 18 months after H. pyloriinoculation, two of five infected animals showed three well-differen

tiated gastric cancers (Fig. 4). Two of the cancers did not developdirectly from intestinal metaplasia but were associated with intestinal

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Fig. 2. Microscopic views of the gastric body of Mongolian gerbils at 18 months afterH. pylori inoculation. A. mucosa is thick with dilated glands. Some dilated glands are seenin Ihe submucosa; however, they have not invaded the proper muscular layer. H&E stain;original magnification, XIO. B. severe infiltration of inflammatory cells. Hyperplasticglands are observed. H&E stain; original magnification. X25.

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Fig. 3. Microscopic views ot the gasine hotly nl M.uvjolun yahiK at IX month-, alterH. pylori inoculation. Marked intestinal metaplasia is seen. Some glands extended into thesubmucosa but not into the proper muscularis layer. A. H&E stain, fi, Alcian blue stain;original magnification. X 10. C. metaplastic glands that are invaded by inflammatory cellsare seen. Inflammatory cell infiltration around dysplastic parts ¡arrows) is more severethan that in another areas. H&E stain; original magnification. X25.

metaplasia at another location (Fig. 4, A-C). The other cancer devel

oped from part of an intestinal metaplasia (Fig. 4D). Poorly differentiated gastric cancer was not observed.

Discussion

This study demonstrates gastric carcinogenesis of H. pylori in aMongolian gerbil model. H. pylori infection first caused gastritis withhyperplastic glands of antrum. Some of these glands easily extended

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Fig. 4. /* and fl, microscopic views of the gastric mucosa of Mongolian gerbils at 18 months after H. pylori inoculation. Well-differentiated adenocarcinoma extended to the muscular layer.Atypical glands and nuclei and abnormal mitosis are seen. A. H&E stain: original magnification. X10. B, H&E stain; original magnification. X50. C, microscopic views of well-differentiatedadenocarcinoma of Mongolian gerbils at 18 months after H. pylori inoculation. The proper muscular layer has been invaded by various sizes of glands with back-to-back appearance. H&E stain;original magnification, X50. D, microscopic views of well-differentiated adenocarcinoma of Mongolian gerbils at 18 months after H. i>\lori inoculation. Atypical glands with atypical nucleiare seen in a metaplaslic gland. This lesion seems to be an early stage of gastric cancer because it has not yet invaded the proper muscular layer. H&E stain; original magnification. X50).

to submucosa by destroying the lamina muscularis mucosa, as reported previously (4, 5). However, they did not invade proper muscularis layer. Therefore, these lesions were not diagnosed as gastriccancer. After that, glands of proper mucosal layer reduced gradually,and some developed intestinal metaplasia.

Atrophie gastritis and intestinal metaplasia appeared in the lessercurvature of the antral mucosa and extended to the body and greatercurvature following inflammation. This agrees with reports that chronicatrophie gastritis and intestinal metaplasia are related to H. p\lori infection (8) and that atrophy or intestinal metaplasia occurs at the lessercurvature and extends to greater curvature in the human (9, 10). Althoughsome animals infected with H. pylori showed atrophie gastritis (11-13),intestinal metaplasia did not appear. A long-term follow-up study onatrophie gastritis in H. /jy/wri-infected Japanese monkeys showed neither

intestinal metaplasia nor gastric cancer (13). A model of Mongoliangerbils infected with H. pylori has been the only animal model forintestinal metaplasia, and it is similar to human gastric lesions caused byH. pylori infection. Intestinal metaplasia increased gradually, and dyspla-sia and well-differentiated gastric cancer occurred in this model. These

consecutive changes support a pathway from H. pylori infection tointestinal-type gastric cancer in humans (6).

Here, two pathways for the occurrence of gastric cancer were seen:first, direct development from intestinal metaplasia, and second, indirect development from intestinal metaplasia, with intestinal meta

plasia existing near the cancer. It is suggested that the two pathwaysexist between atrophie gastritis and well-differentiated gastric cancer.

Diffuse-type gastric cancer did not appear. However, these animalswere infected with the ATCC-43504 strain at 5 weeks old and kept in

the same environment. In humans, H. pylori strain, immune responseof the host, and environment may differ. Further examination usingvarious H. pylori strains and further studies of infection of H. pyloriinfection at various ages are needed.

On the other hand, gastric ulcers were continuously observedthroughout the entire observation period and were localized in area ofpyloric gland, as reported previously (4, 5). This suggests that gastriculcer continues until eradication of H. pylori as well as H. pylori-

indueed gastric ulcers.This study shows gastric carcinogenesis in a H. pylori-infected

Mongolian gerbil model and confirms the IARC/WHO report that H.pylori is "a definite human gastric carcinogen" (14). In addition, this

model is useful for the study of the mechanism of gastric carcinogenesis in H. p\lori infection.

Acknowledgments

We thank Dr. Pelayo Correa for pathological advice and critical reading of

the manuscript. We also thank Mami Kimoto and Kiyomi Ohno for theirtechnical assistance and Dr. Akira Moriuchi for pathological assistance.

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Helicobacter pytori-INDUCED GASTRIC CANCER

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