To investigate the nature of the link between Helicobacter pylori (Hp) infection and stomach carcinogenesis, a study of the glandular stomach of Mongolian gerbils (MGs) was performed. MGs were treated with N-methyl-N-nitrosourea(MNU), followed by inoculation with Hp (groups 1 and 2) or without Hp(group 3), or infected with Hp (groups 4 and 5) or inoculation without Hp (group 6) followed by MNU administration. At week 21, the animals in groups 2 and 5 underwent an eradication procedure. At week 50, the incidences of adenocarcinomas in group 1 (15 of 23) and group 4 (9 of 26) were significantly higher than in group 3 (1 of 15) and group 6 (1 of 18), respectively. Moreover, those in group 2 (5 of 24) and group 5(2 of 22) were lower than in groups 1 and 4, respectively. This study shows that Hp eradication may be useful as a prevention approach against stomach cancer.

From epidemiological findings, there is little a room for doubt that Hp3infection has “positive correlation” with stomach cancer development (1, 2, 3, 4). To demonstrate a “causal link”between Hp infection and stomach carcinogenesis, as well as for intervention studies, it is essential to establish an experimental animal model. We have demonstrated that Hp infection enhances glandular stomach carcinogenesis using MGs. All histological types of stomach cancer are increased on treatment with chemical carcinogens, mimicking human cases (5, 6). The present study was performed to explore the influence of eradication measures on stomach carcinogenesis induced by MNU administration and Hp infection in MGs.

MNU (Sigma Chemical Co., St. Louis, MO) was dissolved in distilled water at concentrations of 30 or 10 ppm and freshly prepared three times per week. The solutions were given as drinking water in light-shielded bottles ad libitum. Hp (ATCC 43504,Rockville, MD) was inoculated on Brucella agar plates(Becton Dickinson and Company, Cockeysville, MD) containing 7% v/v heat-inactivated fetal bovine serum and incubated at 37°C under microaerobic conditions using Anaero Pack Campylo (Mitsubishi Gas Chemical Co., Inc., Tokyo, Japan) at high humidity. Two days later, the bacteria grown on the plates were introduced into Brucellabroth (Becton Dickinson and Company) supplemented with 7% v/v heat-inactivated fetal bovine serum and incubated under the same conditions for 24 h. Samples containing about 1.0 × 108 colony-forming units (0.8 ml) per milliliter were used as the inoculum, delivered i.g. using an oral catheter after fasting for 24 h. For eradication of Hp, a“triple therapy” was performed. The drugs lansoprazole, amoxicillin and clarithromycin were suspended in 0.5% w/w carboxymethyl cellulose sodium salt solution and administered i.g. twice a day for two days at doses of 10, 3, and 30 mg/kg body weight, respectively.

A total of 158 specific pathogen-free male, 7-week-old MGs(Meriones unguiculatu; MGS/Sea, Seac Yoshitomi, Ltd.,Fukuoka, Japan), were housed in steel cages on hardwood chip bedding in an air-conditioned biohazard room (for infection) with a 12-h light/12-h dark cycle. They were given food (Oriental MF; Oriental Yeast Co., Tokyo, Japan) irradiated with 30 kGy gamma rays and autoclaved distilled water ad libitum. The experimental design described below was approved by the Animal Care Committee of the Aichi Cancer Center Research Institute. The animals were treated as follows. In experiment I, 79 gerbils were divided into three groups. They were given MNU in their drinking water at the concentration of 30 ppm for alternate weeks for a total of 5 weeks exposure. On completion of this regimen, they were given autoclaved distilled water; 1 week thereafter, Hp were inoculated (groups 1 and 2, n = 32 and 27, respectively). Group 3 (n = 20) received Brucella broth without Hp. At week 30, five animals in group 1 were killed to check for bacterial infection, and at week 21, all animals in group 2 underwent treatment for eradication. In experiment II, 79 gerbils were divided into three groups. One week after inoculation of Hp (groups 4 and 5, n = 32 and 27, respectively) or the Brucella broth vehicle alone(group 6; n = 20), they were given MNU in their drinking water at the concentration of 10 ppm for 20 weeks continuously. At week 30, five animals in group 4 were killed to check for bacterial infection, and at week 21, all animals in group 5 underwent Hp eradication treatment (Fig. 1). At the 50th experimental week, after 24 h fasting, all animals were subjected to deep ether anesthesia, laparotomized, and exsanguinated from the inferior vena cava, with excision of their stomachs. Tissues were processed for microbiological and histopathological examinations.

For detection of Hp infection, ∼30-mm2 samples of stomach mucosa from the greater curvature, containing both fundic and pyloric glands, were homogenized with 1 ml of Brucellabroth and used for culture of Hp. Aliquots of 100 μl were then inoculated on segregating agar plates for Hp (Eiken Chemical Co.,Tokyo, Japan) and incubated at 37°C under microaerobic conditions for 6 days. Anti-Hp antibodies were measured as described earlier(5). Briefly, blood samples containing a small amount of EDTA were centrifuged at 8000 rpm for 5 min to isolate sera, which were then stored at −80°C. Using the MG sera thus obtained, anti-Hp IgG antibody (GAP-IgG; Biomerica, Newport Beach, CA) was measured by ELISA. The antibody titers were expressed using an A.I. The excised stomachs were fixed in 4% paraformaldehyde in PBS (pH 7.2) or 95% ethanol containing 1% acetic acid, cut into ∼16 strips, processed by standard methods, and embedded in paraffin. Tissues were sectioned at 5μm for staining with H&E, alcian blue-periodic acid-Schiff stain and by immunohistochemistry for Hp (anti-Hp serum; DAKO, Copenhagen,Denmark). Adenocarcinomas of the glandular stomach were classified into well-differentiated lesions, characterized by tubular structures with cellular atypia, poorly differentiated tumors, characterized by little tendency to form glandular structures with severe cellular atypia, and signet ring cell carcinomas, characterized by isolated tumor cells containing abundant amounts of mucin (7).

The two-tailed t test or the Mann-Whitney U test was applied to establish the significance of differences in body weight distributions and titers of anti-Hp antibodies. Survival curves were calculated by the Kaplan-Meier method, and the differences were evaluated using the log-rank test. The adenocarcinoma incidences were assessed by the Fisher’s exact probability method. Ps<0.05 were considered to be statistically significant.

Survival rates for each group were >75%, with no differences among groups by the log-rank test. The body weights in groups 1–6 at experimental week 50 were 71.9 ± 3.5, 83.4 ± 3.0, 85.3 ± 3.6, 92.3 ± 4.2,96.7 ± 2.8, and 103.1 ± 3.0 g(average ± SE), respectively, with the value for group 1 being significantly lower than those for groups 2 and 3(P < 0.05). Hp were detected by culture in all MGs in groups 1 and 4 at week 30. Hp were also detected in all MGs in groups 1 and 4 at week 50. In group 5, 2 of 24 animals were Hp positive by culture at week 50, and these were excluded from later analyses. Other animals in group 5 and all animals in groups 2, 3, and 6 were negative for culture. Titers of anti-Hp antibodies for animals in groups 1, 2, and 3 were 136.1 ± 23.0, 11.4 ± 2.3, and 2.3 ± 0.6 (A.I.; average ± SE), respectively. There are significant differences between each pair (P < 0.001). Values for groups 4,5, and 6 were 466.7 ± 65.1, 100.6 ± 32.4, and 2.5 ± 0.4 (A.I.), respectively, again with significant intergroup differences (P < 0.001).

Histologically, in all MGs in groups 1 and 4, the glandular stomach epithelium showed hyperplastic changes with variable degrees of multifocal cystic dilation and erosion. There was marked infiltration,predominantly of lymphocytes and some macrophages as well as neutrophils in the lamina propria and submucosa, with frequent formation of lymphoid follicles. Intestinal metaplasia was also noted in 23 among 24 gerbils in group 1 and in 25 among 26 animals in group 4. Intestinal metaplasia was not observed in any but the 2 animals that failed to be eradicated for Hp in group 2 and was lacking in groups 3,5, and 6. The incidences of adenocarcinomas of the glandular stomach are summarized in Table 1. Tumors were mostly found in the antral mucosa adjacent to the oxyntic region and comprised well-differentiated (Fig. 2) and poorly differentiated adenocarcinomas (Fig. 3,a), as well as signet ring cell carcinomas (Fig. 3 c). The incidences of adenocarcinomas in group 1 was significantly higher than in group 2 (P < 0.01) and group 3 (P < 0.001). The incidence in group 4 was also significantly higher than those in groups 5 and 6(P < 0.05).

Our present study provides direct evidence that Hp eradication may be useful as a prevention approach. This is the first report of such eradication leading to prevention, to our knowledge. Recently, many clinicians have been prescribing Hp eradication for medical care of patients not only suffering from peptic ulcers (8) and MALT lymphomas (9) but also dyspepsia (10). However, after complete clearance of the bacteria, reflux esophagitis often occurs (11), and this side effect is thought to be an important risk factor for esophageal adenocarcinoma development(12). Therefore, establishment of criteria for Hp eradication is now a top priority.

The most important factors that may influence stomach carcinogenesis are strain diversities of Hp and host reactions. Concerning Hp strains,investigators have studied Cag A and Vac Aintensively. Tomb et al.(13) elucidated the complete Hp genome sequence in 1997, and a full understanding of the pathogenicity of Hp itself should be generated in the near future.

Concerning host factors, we have emphasized the stomach mucosal condition and serological response of the host (6). One interesting point in the MG model is the persistent Hp infection in the stomach, not found in mice and rats. A decrease in the rate of Hp infection with aging and with stomach mucosal damage in MGs and increase in Hp infection related to stomach mucosal damage in mice(14) imply important roles for mucosal conditions of the stomach with regard to Hp infection. T helper 1 cellular immune responses also contribute to Helicobacter-associated gastritis in mice and humans (15), and D’Elios et al.(16) showed that Hp-specific T helper 1 effectors may play a role in generation of peptic ulcers in humans. In our previous study, the titers of anti-Hp antibodies in tumor-bearing animals were found to be higher than in those which were tumor free treated in the same manner (6). This implies that the T helper 2 humoral response may play an important role in stomach carcinogenesis.

For application of Hp eradication to human, timing will presumably be of essential significance. In our present study, we eradicated Hp in the period of active chronic gastritis but before intestinal metaplasia or atrophic gastritis had appeared. The early phase might be expected to be most amenable to intervention. To explore the effectiveness of Hp eradication in later phases, we are now modifying our protocol to clarify what lesions occurring in the Hp-infected stomach are irreversible, true neoplastic changes.

MGs resemble humans in their susceptibility and response to Hp infection (17). It has already been reported that similar pathological changes occur in the glandular stomachs of humans and MGs in response to Hp. In this study, we observed signet ring cell carcinomas as well as poorly differentiated adenocarcinomas and well-differentiated adenocarcinomas, whereas only well-differentiated adenocarcinomas were observed in the stomachs of MGs infected with Hp without any chemical carcinogen exposure (18). This variety of induced stomach cancers as well as the similarity in response to Hp infection suggest advantages of this model for research on stomach carcinogenesis in humans. A recent meta-analysis indicated that Hp infection is equally associated with intestinal and diffuse types of gastric cancer (19), in agreement with our findings.

Using the present carcinogenesis model and adding eradication at various periods, sequential histopathological and molecular biological examinations should allow detailed assessment of the risk of Hp infection in terms of gastric neoplasia and elucidation of the underlying mechanisms.

Fig. 1.

Protocol of the study. ▾, Hp (i.g.); ▿, Brucella broth (i.g.); E, eradication;, MNU in drinking water at the concentration of 30 ppm (dark shading) for alternate weeks in groups 1–3 and at the concentration of 10 ppm (light shading) continuously in groups 4–6.

Fig. 1.

Protocol of the study. ▾, Hp (i.g.); ▿, Brucella broth (i.g.); E, eradication;, MNU in drinking water at the concentration of 30 ppm (dark shading) for alternate weeks in groups 1–3 and at the concentration of 10 ppm (light shading) continuously in groups 4–6.

Close modal
Fig. 2.

Histological sections of gastric mucosa from animals at week 50. Note the well-differentiated glandular stomach adenocarcinoma in a group 1 gerbil (H&E; ×20).

Fig. 2.

Histological sections of gastric mucosa from animals at week 50. Note the well-differentiated glandular stomach adenocarcinoma in a group 1 gerbil (H&E; ×20).

Close modal
Fig. 3.

Histological sections of gastric mucosa from animals at week 50. a, a poorly differentiated adenocarcinoma(alcian blue-periodic acid-Schiff stain; ×20). b, a vessel invasion of adenocarcinoma (H&E; ×200). c, a signet ring cell carcinoma (H&E; ×200).

Fig. 3.

Histological sections of gastric mucosa from animals at week 50. a, a poorly differentiated adenocarcinoma(alcian blue-periodic acid-Schiff stain; ×20). b, a vessel invasion of adenocarcinoma (H&E; ×200). c, a signet ring cell carcinoma (H&E; ×200).

Close modal

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 with 18 U.S.C. Section 1734 solely to indicate this fact.

1

This work was supported in part by a Grant-in-Aid for the Second-term Comprehensive 10-year Strategy for Cancer Control from the Ministry of Health and Welfare, Japan, and by a Grant-in-Aid from the Ministry of Education, Science, Sports and Culture of Japan. During the performance of this work, N. S. was an awardee of a Research Resident Fellowship from the Foundation for the Promotion of Cancer Research.

3

The abbreviations used are: Hp, Helicobacter pylori; MG, Mongolian gerbil; MNU, N-methyl-N-nitrosourea; A.I., arbitrary index; i.g., intragastric.

Table 1

Induction of glandular stomach adenocarcinomas with MNU, Hp infection,and Hp eradication

Group TreatmentaEffective no. of miceNo. of tumor-bearing mice (%)No. of cancerb
WellPoorSig
1c MNU→Hp 23 15 (65.2) 11  
2 MNU→Hp→Er 24 5 (20.8)d  
3 MNU→Br 15 1 (6.7)e  
4 Hp→MNU 26 9 (34.6)  
5 Hp→MNU→Er 22 2 (9.1)f  
6 Br→MNU 18 1 (5.6)f  
Group TreatmentaEffective no. of miceNo. of tumor-bearing mice (%)No. of cancerb
WellPoorSig
1c MNU→Hp 23 15 (65.2) 11  
2 MNU→Hp→Er 24 5 (20.8)d  
3 MNU→Br 15 1 (6.7)e  
4 Hp→MNU 26 9 (34.6)  
5 Hp→MNU→Er 22 2 (9.1)f  
6 Br→MNU 18 1 (5.6)f  
a

Hp, Hp (i.g.); Br, Brucella broth (i.g.);Er, eradication.

b

Well, well-differentiated adenocarcinoma; Poor, poorly differentiated adenocarcinoma; Sig, signet ring cell carcinoma.

c

Four animals in group 1 demonstrated double cancers.

d

P < 0.01 versus group 1 by Fisher’s exact test.

e

P < 0.001 versus group 1 by Fisher’s exact test.

f

P < 0.05 versus group 4 by Fisher’s exact test.

Lansoprazole, amoxicillin, and clarithromycin were kindly provided by Takeda Chemical Industries, Ltd., Kyowa Hakko Kogyo Co.,Ltd., and Taisho Pharmaceutical Co., Ltd., respectively.

1
Nomura A., Stemmermann G. N., Chyou P. H., Kato I., Perez-Perez G. I., Blaser M. J. Helicobacter pylori infection and gastric carcinoma among Japanese Americans in Hawaii.
N. Engl. J. Med.
,
325
:
1132
-1136,  
1991
.
2
Parsonnet J., Friedman G. D., Vandersteen D. P., Chang Y., Vogelman J. H., Orentreich N., Sibley R. K. Helicobacter pylori infection and the risk of gastric carcinoma.
N. Engl. J. Med.
,
325
:
1127
-1131,  
1991
.
3
Forman D., Newell D. G., Fullerton F., Yarnell J. W., Stacey A. R., Wald N., Sitas F. Association between infection with Helicobacter pylori and risk of gastric cancer: evidence from a prospective investigation.
Br. Med. J.
,
302
:
1302
-1305,  
1991
.
4
IARC Working Group on the Evaluation of Carcinogenic Risks to Humans. Helicobacter pylori. In: Schistosomes, Liver Flukes and Helicobacter pylori: Views and Expert Opinions of IARC Working Group on the Evaluation of Carcinogenic Risks to Humans. Lyon: IARC, pp. 177–240, 1994.
5
Sugiyama A., Maruta F., Ikeno T., Ishida K., Kawasaki S., Katsuyama T., Shimizu N., Tatematsu M. Helicobacter pylori infection enhances N-methyl-N-nitrosourea-induced stomach carcinogenesis in the Mongolian gerbil.
Cancer Res.
,
58
:
2067
-2069,  
1998
.
6
Shimizu N., Inada K., Nakanishi H., Tsukamoto T., Ikehara Y., Kaminishi M., Kuramoto S., Sugiyama A., Katsuyama T., Tatematsu M. Helicobacter pylori infection enhances glandular stomach carcinogenesis in Mongolian gerbils treated with chemical carcinogens.
Carcinogenesis (Lond.)
,
20
:
669
-676,  
1999
.
7
Tatematsu M., Yamamoto M., Shimizu N., Yoshikawa A., Fukami H., Kaminishi M., Oohara T., Sugiyama A., Ikeno T. Induction of glandular stomach cancers in Helicobacter pylori-sensitive Mongolian gerbils treated with N-methyl-N-nitrosourea and N-methyl-N′-nitro-N-nitrosoguanidine in drinking water.
Jpn. J. Cancer Res.
,
89
:
97
-104,  
1998
.
8
Peek R. M., Jr., Blaser M. J. Pathophysiology of Helicobacter pylori-induced gastritis and peptic ulcer disease.
Am. J. Med.
,
102
:
200
-207,  
1997
.
9
Neubauer A., Thiede C., Morgner A., Alpen B., Ritter M., Neubauer B., Wundisch T., Ehninger G., Stolte M., Bayerdorffer E. Cure of Helicobacter pylori infection and duration of remission of low-grade gastric mucosa-associated lymphoid tissue lymphoma.
J. Natl. Cancer Inst.
,
89
:
1350
-1355,  
1997
.
10
Agreus L., Talley N. J. Dyspepsia: current understanding and management.
Annu. Rev. Med.
,
49
:
475
-493,  
1998
.
11
Labenz J., Blum A. L., Bayerdorffer E., Meining A., Stolte M., Borsch G. Curing Helicobacter pylori infection in patients with duodenal ulcer may provoke reflux esophagitis.
Gastroenterology
,
112
:
1442
-1447,  
1997
.
12
Naef A. P., Savary M., Ozzello L. Columnar-lined lower esophagus: an acquired lesion with malignant predisposition. Report on 140 cases of Barrett’s esophagus with 12 adenocarcinomas.
J. Thorac. Cardiovasc. Surg.
,
70
:
826
-835,  
1975
.
13
Tomb J. F., White O., Kerlavage A. R., Clayton R. A., Sutton G. G., Fleischmann R. D., Ketchum K. A., Klenk H. P., Gill S., Dougherty B. A., Nelson K., Quackenbush J., Zhou L., Kirkness E. F., Peterson S., Loftus B., Richardson D., Dodson R., Khalak H. G., Glodek A., McKenney K., Fitzgerald L. M., Lee N., Adams M. D., Hickey E. K., Berg D. E., Gocayne J. D., Utterback T. R., Peterson J. D., Kelley J. M., Cotton M. D., Weidman J. M., Fujii C., Bowman C., Watthey L., Wallin E., Hayes W. S., Borodovsky M., Karp P. D., Smith H. O., Fraser C. M., Venter J. C. The complete genome sequence of the gastric pathogen Helicobacter pylori.
Nature (Lond.)
,
388
:
539
-547,  
1997
.
14
Shimizu N., Kaminishi M., Tatematsu M., Tsuji E., Yoshikawa A., Yamaguchi H., Aoki F., Oohara T. Helicobacter pylori promotes development of pepsinogen-altered pyloric glands, a preneoplastic lesion of glandular stomach of BALB/c mice pretreated with N-methyl-N-nitrosourea.
Cancer Lett.
,
123
:
63
-69,  
1998
.
15
Bamford K. B., Fan X., Crowe S. E., Leary J. F., Gourley W. K., Luthra G. K., Brooks E. G., Graham D. Y., Reyes V. E., Ernst P. B. Lymphocytes in the human gastric mucosa during Helicobacter pylori have a T helper cell 1 phenotype.
Gastroenterology
,
114
:
482
-492,  
1998
.
16
D’Elios M. M., Manghetti M., De Carli M., Costa F., Baldari C. T., Burroni D., Telford J. L., Romagnani S., Del Prete G. T helper 1 effector cells specific for Helicobacter pylori in the gastric antrum of patients with peptic ulcer disease.
J. Immunol.
,
158
:
962
-967,  
1997
.
17
Hirayama F., Takagi S., Yokoyama Y., Iwao E., Ikeda Y. Establishment of gastric Helicobacter pylori infection in Mongolian gerbils.
J. Gastroenterol.
,
31
:
24
-28,  
1996
.
18
Honda S., Fujioka T., Tokieda M., Satoh R., Nishizono A., Nasu M. Development of Helicobacter pylori-induced gastric carcinoma in Mongolian gerbils.
Cancer Res.
,
58
:
4255
-4259,  
1998
.
19
Huang J. Q., Sridhar S., Chen Y., Hunt R. H. Meta-analysis of the relationship between Helicobacter pylori seropositivity and gastric cancer.
Gastroenterology
,
114
:
1169
-1179,  
1998
.