Effect of Long-term Administration of Ammonia Water on Rat Gastric Mucosa Combined Effect of Gastric Mucosal Protective Agents

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1 69 Original Article Effect of Long-term Administration of Ammonia Water on Rat Gastric Mucosa Combined Effect of Gastric Mucosal Protective Agents Takao NOGUCHI, Eiji UMEGAKI, Chikao SHIMAMOTO and Ken-ichi KATSU Department of Internal Medicine II, Osaka Medical College, Takatsuki-city, Osaka , Japan Key Words ammonia, Helicobacter pylori, gastric mucus, gastric atrophy, Geranylgeranylacetone(GGA) ABSTRACT We focused on the fact that ammonia induces gastric mucosal damage by Helicobacter pylori, and investigated the effect of ammonia water administration in two kinds of concentration of rat gastric mucosa, by allowing rats free access to ammonia water for 6 weeks. No atrophy developed in the rat gastric mucosa and mucus was maintained in the low concentration group of 0.02% ammonia water, while atrophy was observed in the antral mucosa after 4 weeks and in the body mucosa after 6 weeks of administration in the high concentration group of 0.1% ammonia water. The course of atrophy was similar to that of human atrophic gastritis due to H. pylori infection. Furthermore, both superficial and deep mucus significantly decreased. On the contrary, coadministration of geranylgeranylacetone (GGA), one of gastric mucosal protective agents, inhibited decrease in gastric mucus as well as gastric mucosal atrophy in the 0.1% ammonia water group. In that way, myeloperoxidase (MPO) activity, which is an index of tissue disorders in gastric mucosa, and lipid peroxidation (LPO) activity were significantly increased due to access to ammonia water, while GGA suppressed these activities. It was suggested that gastric mucus may act as a radical scavenger to prevent gastric mucosal damage by ammonia. Introduction In 1982, Warren and Marshall (Australia) discovered Helicobacter pylori (H. pylori). Thereafter, it has been widely accepted that H. pylori induce gastric mucosal damage by a combination of factors, including secreted toxins such as ammonia, enzyme, and outer membrane protein. (1)~(4) Ammonia, especially from urea, plays a major role in gastric mucosal damage by H. pylori, and ammonia alone also triggers gastric mucosal damage. (5) In an H. pylori infected human stomach, the ammonia concentration in gastric juice is approximately 0.01% to 0.02%. (6) Prolonged chronic inflammation for several decades caused by the infection may lead to chronic atrophic gastritis. Ammonia in gastric juice may also contribute to the process. In this study, we continuously administered different concentrations of ammonia water to rats for 6 weeks and investigated the effect on gastric mucosa. We also studied the effects of Address correspondence to: Eiji Umegaki, M.D., PhD., Second Department of Internal Medicine, Osaka Medical College, 2-7 Daigaku-machi, Takatsuki-city, Osaka , Japan Phone: Fax: in2038@poh.osaka-med.ac.jp

2 70 Takao NOGUCHI et al. coadministration of Geranylgeranylacetone (GGA), one of the gastric mucosal protective agents, on gastric mucosal damage by ammonia water. Materials and Methods Experiment 1: Gastric mucosal damage by long-term administration of ammonia water Animals A total of 35 Wister male rats aged 6 weeks and weighing approximately 200 to 250 g (Clea Japan, Inc., Japan) were used in the experiment. a. Control group (control): 5 untreated rats as a control were sacrificed and their stomachs were removed. b. The 0.02% group (0.02% ammonia): 15 rats, divided into 3 groups of 5 each, were allowed free access to 0.02% ammonia water. c. The 0.1% group (0.1% ammonia): 15 rats, divided into 3 groups of 5 each, were allowed free access to 0.1% ammonia water. They were sacrificed and their stomachs were removed 2, 4, and 6 weeks later, respectively. The removed organs were fixed in 10% neutral buffered formalin fixative for 24 hours at 4 C and histological specimens of the antrum and the body were prepared. Atrophy of gastric mucosa The thickness of the gastric mucosa stained by hematoxylin-eosin (HE) was measured, and the ratio of proper gastric glands to the total thickness was defined as atrophy index (%). The thickness was measured at 3 sites including the antrum and the body, and the averages were compared. Mucus in gastric mucosa Both superficial and deep mucus were stained with periodic acid Schiff (PAS) and paradoxical concanavalin A (PCA) staining (7)(8), which was proposed by Ota and Katsuyama, respectively. Area ratio (%) of the area of each positively stained mucus to that of the lamina propria was calculated using a video image processor (9), and defined as a measure of the amount of each mucus. Cell proliferation of gastric mucosa Proliferative cell nuclear antigen (PCNA) immunostaining was performed with labeled streptavidin biotin (LSAB) using an anti-pcna antibody (PC-10, DAKO corporation, USA), and peroxidase was stained with diaminobenzidine (DAB). Positively stained granules or diffuse staining in the cell nucleus was considered positive. The localization and number of PCNApositive cells in the lamina propria mucosa was observed in one field under light microscopy ( 200 magnification) at three sites in the histologic sections of the antrum and the body. The resulting average was defined as the PCNA-positive rate and compared across the group. Experiment 2: Combined effect of gastric mucosa protective agents Animals A total of 24 Wister male rats aged 6 weeks and weighing approximately 200 to 250 g (Clea Japan, Inc., Japan) were used in the experiment. Geranylgeranylacetone (GGA) at 200 mg/kg/day was administered bid using 2% Gum Arabic as a solvent on the rats, which were allowed free access to 0.1% ammonia water for 6 weeks. A single dose of the agent was 0.2 ml. a. Control group (control): This included 8 rats which were administered tap water and 2% Gum Arabic. b. Ammonia group (ammonia): This included 8 rats which were allowed free access to 0.1% ammonia water and administered 2% Gum Arabic. c. GGA group (GGA): This included 8 rats which were allowed free access to 0.1% ammonia water and administered GGA 200 mg/kg/day. They were sacrificed and had their stomachs removed 6 weeks later. Histological specimens of the antrum and body of the posterior wall were prepared, and gastric mucosa was obstained from the anterior wall by blunt dissection and homogenized. Changes in body weight were measured at every week. And, atrophy of gastric mucosa, mucus in gastric mucosa and cell proliferation of gastric mucosa were noted as in experiment 1. Myeloperoxidase (MPO) activity MPO activity was measured to quantitatively evaluate neutrophilic infiltration of the gastric mucosa, and expressed as MPO activity per unit protein. Absorbance was measured at 630 nm after the gastric mucosa was homogenized and tetramethylbenzidine (TMBZ, Dojindo Laboratories, Japan) and H2O2 (Nacalai Tesque, Inc., Japan) were added and allowed to react for 15 minutes (10). At the same time, protein concentration was measured using Bio-Rad Protein Assay (Bio-Rad Laboratories, USA), and the absorbance per unit of protein was calculated. At the initial

3 Effect of ammonia on gastric mucosa 71 measurement, human MPO (Sigma, USA) was used as the reference standard and assayed by the guaiacol method (11). Based on the result, MPO activity (mu/mg) was calculated according to the following formula: MPO activity (mu/mg protein) = absorbance (O.D.) / protein concentration (mg) 1.6 Lipid peroxidation (LPO) activity LPO activity was measured using a LPO-586 kit (BIOXYTECH, France). Statistical analysis: The statistical differences of all data was determined by the Student s t-test and an analysis of variance. All values are expressed as mean SD, and the statistical significance was set at P<0.05 Results Experiment 1: Gastric mucosal damage by long-term administration of ammonia water Atrophy of gastric mucosa In the 0.1% group, the thickness of antral mucosa significantly decreased to 448 ± 18.5 µm after 4 weeks and 454 ± 6.9 µm after 6 weeks (p<0.005%) compared with the control group (523 ± 5.4 µm). The thickness of the body also significantly decreased to 668 ± 21.9 µm after 6 weeks (p<0.005%). However, no significant change of the gastric mucosal thickness was noted throughout the study in the 0.02% group (Figure 1, Table 1). The atrophy index in the antrum of the 0.1% group decreased significantly to 0.46 ± 0.06 after 4 weeks and 0.47 ± 0.04 after 6 weeks (p<0.005%) compared with the control group (0.62 ± 0.06), which indicated a decrease in proper gastric glands. In the 0.02% group, no change in the atrophy index was noted in either the antrum or body (Table 2). Table 1. The effect of long term administration of ammonia on mucosal thickness Table 2. The effect of long term administration of ammonia on atrophy index

4 Takao NOGUCHI et al. 72 Mucous amount in gastric mucosa Superficial mucus which was identified by the PAS staining method increased significantly from 5.3 ± 0.2% at the baseline to 6.4 ± 0.2% after 2 weeks (p<0.005%) in the antrum of the 0.1% group, but significantly decreased to 4.2 ± 0.6% after 4 weeks and 4.6 ± 0.4% after 6 weeks (p<0.01%). There was no significant change in the body of the 0.1% group. In addition, deep mucus which was identified by PCA staining, decreased significantly to 12.0 ± 0.9% after 4 weeks and 11.6 ± 1% after 6 weeks (p<0.005%) in the antrum of the 0.1% group compared with the control group (15.7 ± 1.3%), but there were no significant changes in the body of the 0.1% group (Table 3a,3b). Control Cell proliferation of gastric mucosa Light microscopic findings of PCNA staining were shown in Figure 2. There was no change in the number of PCNA positive cells either in the antrum or the body in the 0.02% group. The number of PCNA positive cells increased significantly to 198 ± 34.8 after 2 weeks and ± 47.1 after 4 weeks compared with the control group (52.6 ± 5.7) in the antrum of the 0.1% group, and almost recovered to the pretreatment number after 6 weeks. An increase by 98.0 ± 22.4 was noted after 6 weeks in the body (Table 4). 0.1% ammonia (6 weeks after) Fig. 1. Light microscopic findings of HE staining. ( 50) Images were taken from antral mucosa. The mucosa administrated with 0.1% ammonia showed atrophy compared to the control. Fig. 2. Microscopic findings of PCNA staining.( 50) PCNA-positive cells were observed mainly at the proliferative zone of lamina propria. Bulletin of the Osaka Medical College , 2007

5 Effect of ammonia on gastric mucosa 73 Table 3a. The effect of long term administration of ammonia on PAS positive mucous contents Table 3b. The effect of long term administration of ammonia on PCA positive mucous contents Table 4. The effect of long term administration of ammonia on PCNA positive cells

6 74 Takao NOGUCHI et al. Experiment 2: Combined effect of gastric mucosa protective agents Change of body weight The rat weight increased over time in all groups, and no significant difference was noted in any groups (Figure 3). Atrophy of gastric mucosa The thickness of antral mucosa decreased significantly to 457 ± 12.7 µm in the ammonia group compared with the control group (551 ± 21.0 µm) after administration of ammonia water(p<0.005), but there were no significant differences between the GGA group and the control group. No change in the body was noted in any groups (Table 5). Atrophy index in the antrum of the ammonia group decreased significantly to 0.56 ± 0.04 compared with 0.65 ± 0.03 in the control group (p<0.005), but the index was 0.69 ± 0.03 in the GGA group. Thus, atrophy of gastric mucosa by ammonia was significantly suppressed (p<0.005). There was no change in the body in any of the groups (Table 6). Fig. 3. The changes of rat body weight after long term administration of ammonia. Table 5. The effect of long term administration of ammonia with GGA on mucosal thickness Table 6. The effect of long term administration of ammonia with GGA on atrophy index

7 Effect of ammonia on gastric mucosa 75 Mucus in gastric mucosa The amount of PAS positive superficial mucus in the antrum decreased significantly to 4.13 ± 0.45 in the ammonia group compared with the control group (5.73 ± 0.46) (p<0.005). However, in the GGA group, the amount of PAS positive superficial mucus was maintained at the same level as that in the control group, and the decrease in superficial mucus caused by ammonia was significantly suppressed (p<0.005). In addition, the amount of PCA positive deep mucus in the antrum decreased significantly to 11.2 ± 0.60 in the ammonia group, compared with 15.1 ± 0.84 in the control group. However, the combined administration of ammonia and GGA significantly suppressed the decrease in deep mucus due to the ammonia (p<0.005). On the other hand, there were no significant changes in PAS positive and PCA positive mucus amounts in the body which had no atrophy of gastric mucosa (Table 7a,7b). Cell proliferation of gastric mucosa The PCNA-positive rate in the antrum was ± 17.8 in the control group. In the ammonia group, the PCNA-positive rate was ± 9.9 and cell proliferation was significantly enhanced (p<0.01). On the other hand, the PCNA-positive rate was 99.0 ± 10.6 in the GGA group, in which no atrophy of gastric mucosa developed, and there was no significant change compared to the control group. No significant differences in PCNApositive rate were noted among 3 groups in the body (Table 8). Table 7a. The effect of long term administration of ammonia with GGA on PAS positive mucous contents Table 7b. The effect of long term administration of ammonia with GGA on PCA positive mucous contents Table 8. The effect of long term administration of ammonia with GGA on PCNA positive cells

8 76 Takao NOGUCHI et al. MPO activity, LPO activity MPO activity, which is an index of inflammatory cellular infiltration and tissue disorders of gastric mucosa, increased significantly to 1.09 ± 0.09 in the ammonia group compared to 0.52 ± 0.27 in the control group (p<0.005). However, in the GGA group, elevation of MPO activity due to ammonia was significantly suppressed to 0.67 ± 0.09 (p<0.005). Like MPO activity, LPO activity, which indicates lipid peroxidation, was 6.6 ± 1.2, 10.5 ± 1.2, and 6.4 ± 1.3 in the control group, the ammonia group, and the GGA group, respectively (Figure 4). Fig. 4. The changes of MPO and LPO activity after long term administration of ammonia with GGA. Discussion Since Palmer s report (12) was published in the 1950 s it has been widely accepted that the stomach is sterile because its strong acid kills bacteria, also, considering human B-type gastritis, so-called chronic atrophic gastritis, develops with ageing. However, the previous established theory was disproved when Warren and Marshall discovered H. pylori and reported the relationship between H. pylori and chronic gastritis (13). Gilvarry, et al. (1) also reported this later on. Now, it is considered that gastric mucosa damage is produced by a combination of monochloramine, including ammonia produced by urease associated with H. pylori infection, secreted toxins such as CagA and VacA (2)(3), enzymes such as mucinase and phospholipase, outer membrane protein like OipA (4), several types of heat shock proteins, and inflammatory cytokines. It is especially well known that ammonia is generated when urease hydrolyzes urea into ammonia and carbon dioxide acting as a catalyst induces gastric mucosal damage alone. (5) The ammonia concentration in Japanese patients infected with H. pylori ranges from 0.01 to 0.02% (6), which may be approximately 3-fold higher than that in non-infected Japanese, with a significant difference. In this 6-week administration of 0.02% ammonia water, no significant change was noted in the amount of gastric mucus, atrophy of gastric mucosa, or cell proliferation of gastric mucosa. Takahashi, et al. (14) reported a significant correlation between gastric ammonia concentration and gastritis. Likewise, the highconcentration of ammonia administration (0.1%) in this experiment induced a decrease in gastric mucus as well as gastric mucosa atrophy over a relatively short time. The administration of 0.1% ammonia water resulted in atrophy of the antrum 4 weeks later and a decrease in the body mucosal thickness 6 weeks later. Such a process is similar to the course of human B-type gastritis, i.e. chronic atrophic gastritis, and suggests that ammonia water administration induces atrophic gastritis. The kinetics of ammonia in the stomach is assumed to be related to why there is a difference in the degree of atrophy in the antrum and the body. In the stomach, ammonia reacts with

9 Effect of ammonia on gastric mucosa 77 hydrochloric acid (HCl) to produce ammonium chloride (NH4Cl). Generally, the ph in the antrum where gastric secretion is low compared to the body is high. In ammonia-induced cytotoxicity, NH3 is more potent than NH4Cl, and in the antrum where the ph is high, cellular damage due to NH3 that has more potent cytotoxicity, was seen early compared to the body. The cell kinetics of gastric mucosa were not changed in the 0.02% ammonia group which showed no mucosal changes. However, mucosal change increased after 2 weeks of 0.1% ammonia water administration. Tsujii, et al. (15) reported that BrdU administration to rats administered with ammonia facilitated cell kinetics in the atrophy of gastric mucosa. Similarly, this experiment indicated that the cell proliferation ability of gastric mucosa as well as cell kinetics was enhanced after 2 and 4 weeks at the early stage of atrophy of gastric mucosa. In terms of gastric mucus, Sugiyama, et al. (16) reported that ammonia administration into the rat stomach decreased PAS positive superficial mucus. As this mechanism of action, gastric mucus dissolving enzyme such as protease produced by H. pylori may destroy the gastric superficial mucous layer. However, the superficial mucus after 2 weeks of 0.1% ammonia water administration increased in this experiment. It is speculated that a cytoprotective mechanism worked because ammonia water temporarily acted on gastric mucosa as a mild irritant. This experiment shows decreasing not only of the superficial layer mucus but also of the deep layer mucus with atrophy of gastric mucosa. Furthermore, we investigated the effect of GGA, a gastric mucosal protective agent and heat shock protein inducer, in order to elucidate the role of gastric mucus in gastric mucosal damage caused by ammonia. GGA is a gastric mucosal protective agent that promotes gastric mucosal synthesis, and increases superficial and deep gastric mucus (17). Atrophy of the antral mucosa and decrease of gastric mucus in the superficial and deep layers were observed when the rats were allowed free access to 0.1% ammonia water for 6 weeks. At the same time, MPO activity, which is an index of tissue disorders caused by neutrophilic infiltration and free radicals (18), as well as LPO activity which indicates lipid peroxidation significantly increased. These changes resulted from gastric mucosal damage (19)(20) by free radicals such as monochloramine produced by ammonia, but no changes were noted in the GGA group. Yanaka et al (21) suggested that gastric mucus acted as a radical scavenger in an in vitro experiment. The present in vivo experiment which succeeded in suppressing gastric mucosal damage by ammonia, shows that gastric mucus may act as a radical scavenger and play a major role in the prevention of gastric mucosal damage. Conclusion This experiment shows that long-term ammonia water administration to rats induces atrophy of gastric mucosa similar to human atrophic gastritis and that gastric mucosal damage caused by ammonia can be used as a model of atrophic gastritis caused by H. pylori infection. It is considered that such changes may be prevented by administration of GGA, a gastric mucosal protective agent, and that maintenance of gastric mucus as a radical scavenger may prevent gastric mucosal damage caused by ammonia. Reference 1. Gilvarry J.M, Leen E, Sweeney E, O morain C.A. The long-term effect of Helicobacter pylori on gastric mucosa. Eur. J.Gastroenterol.Hepatol. 1994;6: Hatakeyama M. Oncogenic mechanisms of the Helicobacter pylori CagA protein. Nat. Rev. Cancer 2004;4-9: Wada A, Yahiro K, Hirayama T. Helicobacter pylori vacuolating cytoxin(vac A) and its modulatoryeffects in host cells. Protein, Nucleic Acid and Enzyme 2001;46-4: (in Japanese) 4. Yamaoka Y, Kwon D.H, Graham D.Y. A M(r) 34,000 proinflammatory outer membrance protein (oipa) of Helicobacter pylori. Proc. Natl. Acad. Sci. USA 2000;97: Kawano S, Tsujii M, Fusamoto H, Sato N, Kamada T. Chonic effect of intragastric ammonia on gastric mucosal structures in rats. Digestive Diseases and Sciences 1991;36: Kochiyama T. Clinical study of Campylobacter pylori in stomach disease. Gastroenterological Endoscopy 1989;31:3-13. (in Japanese) 7. Hayama M, Katsuyama T, Nakayama J, Akamatsu T, Honda T. A combined stain for identifying epithelial cells of the gastric mucosa. Stain Technology 1987;62: Ota H, Katsuyama T, Ishii K, Nakayama J, Shiozawa T, Tsukahara Y. A dual staining

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