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Ammonia: a possible promotor in Helicobacter pylori-related gastric carcinogenesis
15
Cancer Letters, 65 (1992)15- 18 Elsevier Scientific Publishers
Ireland
Ltd.
Ammonia: a possible promotor carcinogenesis
in Helicobacter
pylori-related
gastric
Masahiko Tsujii, Sunao Kawano, Shingo Tsuji, Kouichi Nagano, Toshifumi Hayashi, Hideyuki Fusamoto, Takenobu Kamada and Kazuya Tamura
Ito, Nobuhiko
Department of Medicine, Osaka University Medical School and Division of Gastroenterology, l-1 -50 Fukushima, Fukushima-ku, Osaka (Japan)
National Hospital.
Osaka
(Received 26 November 1991) (Revision received 24 April 1992) (Accepted 27 April 1992)
Summary
Introduction
Helicobacter pylori (HP) has been shown to possibly
HP has
be a pathogen of gastric carcinoma. urease activity and produces ammonia
in the stomach. In this study, the role of ammonia on gastric carcinogenesis induced by Nmethyl-N’-nitro-N-nitrosoguanidine (MNNG) were investigated in rats. Afier 24 weeks pretreatment with MNNG (83 mg/f), 0.01% ammonia or tap water as a drinking water was administered for 24 weeks. The ammoniatreated rats showed a significantly higher incidence of gastric cancer (percent of animals with tumors and number of tumors per rat). Ammonia would thus appear to have an important role in HP-related human gastic carcinogenesis.
Keywords: Helicobacter pylori; ammonia; gastric cancer; atrophic gastritis Correspondence to: Masahiko Tsujii, Department of Medicine, Osaka University Medical School and Division of Gastroenterology, Osaka National Hospital, l-l-50 Fukushima, Fukushima-ku, Osaka, Japan.
0304-3835/92/$05.00
Printed
and Published
0
1992
in Ireland
Elsevier Scientific Publishers
Evidence is accumulating that HP infection plays a major contributory role in epidemic gastric cancer. The investigations examining the correlation between HP infection, as reflected in immunoglobulin G serum antibodies and risk of gastric cancer show that the differences in the infection patterns of HP by socioeconomical class are consistent with patterns of gastric cancer [12] and that the incidence of positive ELISA results was high in patients with gastric cancer [17]. These data suggest that most patients with gastric cancer have been infected with HP at some point in their life. Mechanisms for HP-related carcinogenesis in gastric mucosa are, however, not known. HP is recognized as having a major etiological role in type B chronic atrophic gastritis [8]. In pathogenesis of atrophic gastritis, continuous mucosal cell damage and acceleration of mucosal desquamation is thought to be involved, with consequent stimulation of cellular proliferation in the gastric mucosa, suggesting association with premalignancy [3,4]. As an etiologic factor in HP-related mucosal damage, increasing Ireland Ltd
16
evidence has been collecting of ammonia produced by HP in the stomach. Ammonia concentration of gastric juice in HP-infected subjects is higher than in HP negative subjects (0.015% vs. <0.005%) [lo]. In our previous study, 0.01% ammonia chronically administered caused gastric mucosal atrophy [9] and led to acceleration of cell proliferation [15]. In this study, we examined the promotional effect of chronic administration with 0.01% ammonia on gastric carcinogenesis in rats. Materials and Methods Male Sprague - Dawley (n = 85) rats aged 5 weeks, initially weighing 120 g were given drinking water containing MNNG (Sigma Co., St. Louis) (83 mg/l) for 24 weeks, At week 25, 80 rats were randomly divided into the following two groups: Group 1 were given tap water and group 2 were given 0.01% ammonia solution as the drinking fluid for 24 weeks. The animals which survived more than 45 weeks were included in the effective numbers, because the first cancer of the glandular stomach was found in a rat in group 1, which died at week 45. All rats were autopsied when they become moribund during the experiment or at the end of week 48 and sections of stomach were examined by a pathologist without knowledge as to which group they belonged to. Adenocarcinomas were histologically defined as lesions in which neoplastic glands had penetrated the muscularis mucosa, involving the submucosa or deeper layers of stomach. Data were given as mean f SD and were analyzed by Chi-square test or the unpaired ttest. Statistical significance was declared when a probability value was less than 0.01. Results A total of 76 rats (39 and 37 rats in group 1 and 2, respectively) were used for analysis. Polypoid tumors - rarely with central ulceration - were observed in the glandular stomachs. Microscopically, all tumors were
Table 1. Incidence and number of gastric cancers in
MNNG-treated rats (mean
l
SD).
Groupa
Body wt (g)
No. of rats with gastric cancer (41)
No. of gastric cancer/ tumor-bearing rat
1 2
559 * 73 561 f 86
12/39 (31) 26/37 (70)’
1.3 f 0.6 2.1 zt 1.4’
‘Significantly different from the value for group 1, P < 0.01. “Treatment of group 1: MNNG (24 weeks) + tap water (24 weeks); group 2: MNNG (24 weeks) + 0.01% ammonia (24 weeks).
characterized as intestinal or differentiated and not diffuse or undifferentiated. As shown in Table I, the incidence and number of gastric cancers per tumor-bearing rat were significantly greater in group 2 than in group 1. Seven tumors were found in the fundus of stomach in group 2, while only one tumor was found in the fundus in group 1. None of the animals had florid ulcers or ulcer scars in the glandular stomach. All animals had signs of gastritis, but no intestinal metaplasia was observed. The incidence of small intestinal tumors was comparable for groups 1 and 2 (17/39 and 23/37, respectively) and was apparently not affected by the treatment with 0.01% ammonia. Macroscopic metastases of the liver could be found in 3 rats in group 2, but none in any rats in group 1. There was no significant difference in body weight or daily fluid intake (about 50 ml/day) in the two groups. Discussion There is a strong association between the sequelae of HP gastritis, such as atrophy, intestinal metaplasia and gastric cancer [6,11]. Furthermore, in countries with a high incidence of gastric carcinoma, such as Japan, the age-specific incidence of HP gastritis closely parallels that of gastric carcinoma [5]. Even in other countries, Sitas reported that the differences in the infection patterns of HP by
17
socioeconomical class are consistent with those of gastric cancer 1121 and Wulffen noted the incidence of positive IgG antibody against HP to be high in patients with gastric cancer [17]. HP infection would thus appear to be closely related to human gastric cancer. However, the mechanism is still unclear. A causative role is now accepted for HP in type B chronic atrophic gastritis [8]. Several investigators have reported the higher rate of cell proliferation and the increased rates of epithelial cell exfoliation in patients with atrophic gastritis, suggesting a more rapid turnover of the gastric mucosa [3]. These data suggest that some factors which cause continuous mucosal cell damage and accelerate the mucosal desquamation might be involved in pathogenesis of atrophic gastritis, with consequent stimulation of cellular proliferation, believed as a precancerous condition. It has been recently suggested that ammonia is a major factor causing HP-related gastroduodenal mucosal lesions [lo]. HP has strong urease activity and produces abundant ammonia from urea in the stomach. Ammonia concentration of gastric juice in HP-infected subjects is higher than in HP negative subjects (0.015% vs. < 0.005%) [8]. We have already reported that ammonia (O.Ol- 0.02%) inhibits the mitochondrial and isolated cellular respiration and has cytotoxic effect on gastric mucosal cells [16]. The chronic administration of 0.01% ammonia was found to cause atrophic changes in rat gastric mucosa resembling those in human type B atrophic gastritis [9], leading consequently to accelerated cell proliferation in the gastric mucosa [15], Mucosal cell desquamation and cell proliferation have also been shown to be accelerated in HP-positive subjects and a significant correlation between ammonia concentration in gastric juice and mucosal cell desquamation and cell proliferation has also been shown [ 141. Ammonia produced by Helicobacter pylori may thus possibly accelerate mucosal cell desquamation and stimulate cell proliferation in human as well as animals [15] by a compensatory mechanism. Ammonia may also be directly involved in cell
proliferation. Ammonia is a weak base and non-ionized NH3 easily crosses membranes and reacts to form NH4 + and OH - in cells, with consequent increase in intracellular pH [13]. There is evidence that cytoplasmic pH may make an important contribution to the metabolic activation of various resting cells and growth control [l]. Thus, ammonia may be considered to accelerate gastric mucosal cell proliferation. These conditions may be a prerequisite for gastric carcinogenesis. An investigation was thus made of the role of ammonia in MNNG-induced gastric cancer in rats. The oral administration of 0.01% ammonia following MNNG pretreatment was clearly shown to cause a significant increase in the incidence and number of gastric cancers, indicating ammonia to have a promoting effect in gastric carcinogenesis induced by MNNG. In the present study, more gastric tumors in the fundus of stomach in the ammonia-treated group were found than in control group. Gastrin is an established trophic hormone of gastric oxyntic mucosa [7], although gastrin is reported to exert an inhibitory effect in gastric carcinogenesis in the antral mucosa [2]. Ammonia might neutralize gastric acid and could result in increasing serum gastrin level. Further investigation regarding growth factors, such as gastrin, epidermal growth factor and so on, should be needed for detailed determination of the mechanism of gastric carcinogenesis in this model. The present study shows the chronic oral administration of 0.01% ammonia to exert a promoting effect on rat gastric cancer induced by MNNG. Ammonia may thus make an important contribution to human gastric carcinogenesis in HP-infected patients.
References Busa, W. and Nuccitelli, R. (1984) Metabolic regulation via intracellular pH. Am. J. Physiol., 246, R409 - R438. Casteleyn, P.P., DuBrasquest, M. and Willems, G. (1977) Opposite effects of gastrin on cell proliferation in the antrum and other parts of the upper gastrointestinal tract in the rat. Am. J. Dig. Dis., 22, 798- 804. Deshner, FE., Winawer, S.J. and Lipkin, M. (1972)
18
4
5
6 7
8
9
10
Patterns of nucleic acid and protein synthesis in normal human gastric mucosa and atrophic gastritis. J. Natl. Cancer Inst., 48, 1567 - 1574. Glass, G.B. and Pitchumoni, C.S. (1975) Atrophic gastritis. Structural and ultrastructural alterations, exfoliative cytology and enzyme cytochemistry and histochemistry, proliferation kinetics, immunological derangements and other causes and clinical associations and sequelae. Hum. Pathol., 6, 219-250. Graham, D.Y. and Klein, P.D. (1987) Campylobacter pyloridis gastritis: The past, the present and speculation about the future. Am. J. Gastroenterol., 82, 283 - 286. Green, L.K. and Klein, P.D. (1990) Gastritis in the elderly. Gastroenterol. Clin. N. Am., 19, 273-292. Johnson, L.R. (1981) Regulation of gastrointestinal growth. In: Physiology of Gastrointestinal Tract, pp, 169- 196. Raven, New York. Karnes, W.E., Samloff, I.M., Siurala, M., Kekki, M., Sipponen, P., Kim, W.R. and Walsh, J.H. (1991) Positive antibody and negative tissue staining for serum Helicobacter pylori in subjects with atrophic body gastritis. Gastroenterology, 101, 167 - 174. Kawano, S., Tsujii, M., Fusamoto, H., Sato, N. and Kamada, T. (1991) Chronic effect of intragastric ammonia on gastric mucosal structures in rats. Dig. Dis. Sci., 36, 33-38. Langton, S.R. and Marshall, B.J. (1986) Urea hydrolysis in patients with Campylobacter pyloridis infection. Lancet, i, 965 - 966.
11 12
13
14
15
16
17
Sipponen, P. (1989) Atrophic gastritis as a premalignant condition. Ann. Med., 21, 287-290. Sitas, F., Forman, D., Yarnell, J. W., Burr, M.L., Elwood, P.C., Pedley, S. and Marks, K.J. (1991) Helicobacter pylori infection rates in relation to age and social class in a population of Welsh men. Gut, 32, 25- 28. Townsley, MC., Paradiso, A.M. and Machen, T.E. (1988) Na/H exchange in antral cells isolated from Necturus, rabbit and guinea pig. Gastroenterology, 95, 302 - 310. Tsujii, M., Kawano, S., Ishigami, Y., Nagano, K., Tsuji, S., Hayashi, N., Masuda, E., Fukui, H., Sasayama, Y., Michida, T., Chen, S.S., Fusamoto, H. and Kamada, T. (1991) Ammonia-produced by Helicobacter pylon accelerates cell proliferation rate of human gastric mucosa. Gastroenterology, 100, A176. Tsujii, M., Kawano, S., ho, T., Sasaki, Y., Tsuji, S., Nagano, K., Oshita, M., Matsunaga, T., Hayashi, N., Fusamoto, H. and Kamada, T. (1991) Ammonia increases cell proliferation with progress of atrophic change in rat gastric mucosa. Gastroenterol., 100, Al77 Tsujii, M., Kawano, S., Tsuji, S., Fusamoto, H., Kamada, T. and Sato, N. Mechanism of gastric mucosal damage induced by ammonia. Gastroenterology, in press. Wulffen, H. and Grote, H.J. (1988) Enzyme-linked immunosorbent assay for detection of immunoglobin A and G antibodies to Campylobacter pylori. Eur. J. Clin. Microbial. Infect. Dis., 7, 559 -565.
Cancer Letters, 65 (1992)15- 18 Elsevier Scientific Publishers
Ireland
Ltd.
Ammonia: a possible promotor carcinogenesis
in Helicobacter
pylori-related
gastric
Masahiko Tsujii, Sunao Kawano, Shingo Tsuji, Kouichi Nagano, Toshifumi Hayashi, Hideyuki Fusamoto, Takenobu Kamada and Kazuya Tamura
Ito, Nobuhiko
Department of Medicine, Osaka University Medical School and Division of Gastroenterology, l-1 -50 Fukushima, Fukushima-ku, Osaka (Japan)
National Hospital.
Osaka
(Received 26 November 1991) (Revision received 24 April 1992) (Accepted 27 April 1992)
Summary
Introduction
Helicobacter pylori (HP) has been shown to possibly
HP has
be a pathogen of gastric carcinoma. urease activity and produces ammonia
in the stomach. In this study, the role of ammonia on gastric carcinogenesis induced by Nmethyl-N’-nitro-N-nitrosoguanidine (MNNG) were investigated in rats. Afier 24 weeks pretreatment with MNNG (83 mg/f), 0.01% ammonia or tap water as a drinking water was administered for 24 weeks. The ammoniatreated rats showed a significantly higher incidence of gastric cancer (percent of animals with tumors and number of tumors per rat). Ammonia would thus appear to have an important role in HP-related human gastic carcinogenesis.
Keywords: Helicobacter pylori; ammonia; gastric cancer; atrophic gastritis Correspondence to: Masahiko Tsujii, Department of Medicine, Osaka University Medical School and Division of Gastroenterology, Osaka National Hospital, l-l-50 Fukushima, Fukushima-ku, Osaka, Japan.
0304-3835/92/$05.00
Printed
and Published
0
1992
in Ireland
Elsevier Scientific Publishers
Evidence is accumulating that HP infection plays a major contributory role in epidemic gastric cancer. The investigations examining the correlation between HP infection, as reflected in immunoglobulin G serum antibodies and risk of gastric cancer show that the differences in the infection patterns of HP by socioeconomical class are consistent with patterns of gastric cancer [12] and that the incidence of positive ELISA results was high in patients with gastric cancer [17]. These data suggest that most patients with gastric cancer have been infected with HP at some point in their life. Mechanisms for HP-related carcinogenesis in gastric mucosa are, however, not known. HP is recognized as having a major etiological role in type B chronic atrophic gastritis [8]. In pathogenesis of atrophic gastritis, continuous mucosal cell damage and acceleration of mucosal desquamation is thought to be involved, with consequent stimulation of cellular proliferation in the gastric mucosa, suggesting association with premalignancy [3,4]. As an etiologic factor in HP-related mucosal damage, increasing Ireland Ltd
16
evidence has been collecting of ammonia produced by HP in the stomach. Ammonia concentration of gastric juice in HP-infected subjects is higher than in HP negative subjects (0.015% vs. <0.005%) [lo]. In our previous study, 0.01% ammonia chronically administered caused gastric mucosal atrophy [9] and led to acceleration of cell proliferation [15]. In this study, we examined the promotional effect of chronic administration with 0.01% ammonia on gastric carcinogenesis in rats. Materials and Methods Male Sprague - Dawley (n = 85) rats aged 5 weeks, initially weighing 120 g were given drinking water containing MNNG (Sigma Co., St. Louis) (83 mg/l) for 24 weeks, At week 25, 80 rats were randomly divided into the following two groups: Group 1 were given tap water and group 2 were given 0.01% ammonia solution as the drinking fluid for 24 weeks. The animals which survived more than 45 weeks were included in the effective numbers, because the first cancer of the glandular stomach was found in a rat in group 1, which died at week 45. All rats were autopsied when they become moribund during the experiment or at the end of week 48 and sections of stomach were examined by a pathologist without knowledge as to which group they belonged to. Adenocarcinomas were histologically defined as lesions in which neoplastic glands had penetrated the muscularis mucosa, involving the submucosa or deeper layers of stomach. Data were given as mean f SD and were analyzed by Chi-square test or the unpaired ttest. Statistical significance was declared when a probability value was less than 0.01. Results A total of 76 rats (39 and 37 rats in group 1 and 2, respectively) were used for analysis. Polypoid tumors - rarely with central ulceration - were observed in the glandular stomachs. Microscopically, all tumors were
Table 1. Incidence and number of gastric cancers in
MNNG-treated rats (mean
l
SD).
Groupa
Body wt (g)
No. of rats with gastric cancer (41)
No. of gastric cancer/ tumor-bearing rat
1 2
559 * 73 561 f 86
12/39 (31) 26/37 (70)’
1.3 f 0.6 2.1 zt 1.4’
‘Significantly different from the value for group 1, P < 0.01. “Treatment of group 1: MNNG (24 weeks) + tap water (24 weeks); group 2: MNNG (24 weeks) + 0.01% ammonia (24 weeks).
characterized as intestinal or differentiated and not diffuse or undifferentiated. As shown in Table I, the incidence and number of gastric cancers per tumor-bearing rat were significantly greater in group 2 than in group 1. Seven tumors were found in the fundus of stomach in group 2, while only one tumor was found in the fundus in group 1. None of the animals had florid ulcers or ulcer scars in the glandular stomach. All animals had signs of gastritis, but no intestinal metaplasia was observed. The incidence of small intestinal tumors was comparable for groups 1 and 2 (17/39 and 23/37, respectively) and was apparently not affected by the treatment with 0.01% ammonia. Macroscopic metastases of the liver could be found in 3 rats in group 2, but none in any rats in group 1. There was no significant difference in body weight or daily fluid intake (about 50 ml/day) in the two groups. Discussion There is a strong association between the sequelae of HP gastritis, such as atrophy, intestinal metaplasia and gastric cancer [6,11]. Furthermore, in countries with a high incidence of gastric carcinoma, such as Japan, the age-specific incidence of HP gastritis closely parallels that of gastric carcinoma [5]. Even in other countries, Sitas reported that the differences in the infection patterns of HP by
17
socioeconomical class are consistent with those of gastric cancer 1121 and Wulffen noted the incidence of positive IgG antibody against HP to be high in patients with gastric cancer [17]. HP infection would thus appear to be closely related to human gastric cancer. However, the mechanism is still unclear. A causative role is now accepted for HP in type B chronic atrophic gastritis [8]. Several investigators have reported the higher rate of cell proliferation and the increased rates of epithelial cell exfoliation in patients with atrophic gastritis, suggesting a more rapid turnover of the gastric mucosa [3]. These data suggest that some factors which cause continuous mucosal cell damage and accelerate the mucosal desquamation might be involved in pathogenesis of atrophic gastritis, with consequent stimulation of cellular proliferation, believed as a precancerous condition. It has been recently suggested that ammonia is a major factor causing HP-related gastroduodenal mucosal lesions [lo]. HP has strong urease activity and produces abundant ammonia from urea in the stomach. Ammonia concentration of gastric juice in HP-infected subjects is higher than in HP negative subjects (0.015% vs. < 0.005%) [8]. We have already reported that ammonia (O.Ol- 0.02%) inhibits the mitochondrial and isolated cellular respiration and has cytotoxic effect on gastric mucosal cells [16]. The chronic administration of 0.01% ammonia was found to cause atrophic changes in rat gastric mucosa resembling those in human type B atrophic gastritis [9], leading consequently to accelerated cell proliferation in the gastric mucosa [15], Mucosal cell desquamation and cell proliferation have also been shown to be accelerated in HP-positive subjects and a significant correlation between ammonia concentration in gastric juice and mucosal cell desquamation and cell proliferation has also been shown [ 141. Ammonia produced by Helicobacter pylori may thus possibly accelerate mucosal cell desquamation and stimulate cell proliferation in human as well as animals [15] by a compensatory mechanism. Ammonia may also be directly involved in cell
proliferation. Ammonia is a weak base and non-ionized NH3 easily crosses membranes and reacts to form NH4 + and OH - in cells, with consequent increase in intracellular pH [13]. There is evidence that cytoplasmic pH may make an important contribution to the metabolic activation of various resting cells and growth control [l]. Thus, ammonia may be considered to accelerate gastric mucosal cell proliferation. These conditions may be a prerequisite for gastric carcinogenesis. An investigation was thus made of the role of ammonia in MNNG-induced gastric cancer in rats. The oral administration of 0.01% ammonia following MNNG pretreatment was clearly shown to cause a significant increase in the incidence and number of gastric cancers, indicating ammonia to have a promoting effect in gastric carcinogenesis induced by MNNG. In the present study, more gastric tumors in the fundus of stomach in the ammonia-treated group were found than in control group. Gastrin is an established trophic hormone of gastric oxyntic mucosa [7], although gastrin is reported to exert an inhibitory effect in gastric carcinogenesis in the antral mucosa [2]. Ammonia might neutralize gastric acid and could result in increasing serum gastrin level. Further investigation regarding growth factors, such as gastrin, epidermal growth factor and so on, should be needed for detailed determination of the mechanism of gastric carcinogenesis in this model. The present study shows the chronic oral administration of 0.01% ammonia to exert a promoting effect on rat gastric cancer induced by MNNG. Ammonia may thus make an important contribution to human gastric carcinogenesis in HP-infected patients.
References Busa, W. and Nuccitelli, R. (1984) Metabolic regulation via intracellular pH. Am. J. Physiol., 246, R409 - R438. Casteleyn, P.P., DuBrasquest, M. and Willems, G. (1977) Opposite effects of gastrin on cell proliferation in the antrum and other parts of the upper gastrointestinal tract in the rat. Am. J. Dig. Dis., 22, 798- 804. Deshner, FE., Winawer, S.J. and Lipkin, M. (1972)
18
4
5
6 7
8
9
10
Patterns of nucleic acid and protein synthesis in normal human gastric mucosa and atrophic gastritis. J. Natl. Cancer Inst., 48, 1567 - 1574. Glass, G.B. and Pitchumoni, C.S. (1975) Atrophic gastritis. Structural and ultrastructural alterations, exfoliative cytology and enzyme cytochemistry and histochemistry, proliferation kinetics, immunological derangements and other causes and clinical associations and sequelae. Hum. Pathol., 6, 219-250. Graham, D.Y. and Klein, P.D. (1987) Campylobacter pyloridis gastritis: The past, the present and speculation about the future. Am. J. Gastroenterol., 82, 283 - 286. Green, L.K. and Klein, P.D. (1990) Gastritis in the elderly. Gastroenterol. Clin. N. Am., 19, 273-292. Johnson, L.R. (1981) Regulation of gastrointestinal growth. In: Physiology of Gastrointestinal Tract, pp, 169- 196. Raven, New York. Karnes, W.E., Samloff, I.M., Siurala, M., Kekki, M., Sipponen, P., Kim, W.R. and Walsh, J.H. (1991) Positive antibody and negative tissue staining for serum Helicobacter pylori in subjects with atrophic body gastritis. Gastroenterology, 101, 167 - 174. Kawano, S., Tsujii, M., Fusamoto, H., Sato, N. and Kamada, T. (1991) Chronic effect of intragastric ammonia on gastric mucosal structures in rats. Dig. Dis. Sci., 36, 33-38. Langton, S.R. and Marshall, B.J. (1986) Urea hydrolysis in patients with Campylobacter pyloridis infection. Lancet, i, 965 - 966.
11 12
13
14
15
16
17
Sipponen, P. (1989) Atrophic gastritis as a premalignant condition. Ann. Med., 21, 287-290. Sitas, F., Forman, D., Yarnell, J. W., Burr, M.L., Elwood, P.C., Pedley, S. and Marks, K.J. (1991) Helicobacter pylori infection rates in relation to age and social class in a population of Welsh men. Gut, 32, 25- 28. Townsley, MC., Paradiso, A.M. and Machen, T.E. (1988) Na/H exchange in antral cells isolated from Necturus, rabbit and guinea pig. Gastroenterology, 95, 302 - 310. Tsujii, M., Kawano, S., Ishigami, Y., Nagano, K., Tsuji, S., Hayashi, N., Masuda, E., Fukui, H., Sasayama, Y., Michida, T., Chen, S.S., Fusamoto, H. and Kamada, T. (1991) Ammonia-produced by Helicobacter pylon accelerates cell proliferation rate of human gastric mucosa. Gastroenterology, 100, A176. Tsujii, M., Kawano, S., ho, T., Sasaki, Y., Tsuji, S., Nagano, K., Oshita, M., Matsunaga, T., Hayashi, N., Fusamoto, H. and Kamada, T. (1991) Ammonia increases cell proliferation with progress of atrophic change in rat gastric mucosa. Gastroenterol., 100, Al77 Tsujii, M., Kawano, S., Tsuji, S., Fusamoto, H., Kamada, T. and Sato, N. Mechanism of gastric mucosal damage induced by ammonia. Gastroenterology, in press. Wulffen, H. and Grote, H.J. (1988) Enzyme-linked immunosorbent assay for detection of immunoglobin A and G antibodies to Campylobacter pylori. Eur. J. Clin. Microbial. Infect. Dis., 7, 559 -565.