Effects of butylated hydroxyanisole, butylated hydroxytoluene, and NaCl on gastric carcinogenesis initiated with N-methyl-N'-nitro-N-nitrosoguanidine in F344 rats

Promoting activities of butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), and NaCl and of combinations of these antioxidants with NaCl on gastric carcinogenesis initiated by N-methyl-N'-nitro-N-nitrosoguanidine [(MNNG) (CAS: 70-25-7; 1-methyl-3-nitro-1-nitrosoguanidine] were investigated in male inbred F344 rats. Animals, 6-week old, were given an intragastric administration of MNNG at 150 mg/kg body weight by gastric tube and 1 week later were placed on a diet containing BHA (0.5%), BHT (1.0%), NaCl (5.0%), BHA (0.5%) plus NaCl (5%), or BHT (1.0%) plus NaCl (5.0%) for 51 weeks. Control rats received no further treatment after MNNG administration. A single intragastric application of MNNG to rats induced multiple epithelial tumors of the forestomach and a few epithelial tumors of the glandular stomach after 52 weeks. Squamous cell carcinomas of the forestomach were seen in 2 of 18 effective rats (11.1%) in the control groups, and the incidences in the groups receiving the subsequent treatment were 45.0% with BHA, 15.8% with BHT, 30% with NaCl, 70% with BHA plus NaCl, and 52.9% with BHT plus NaCl. Differences in the incidences of squamous cell carcinoma between the controls and groups given BHA, BHA plus NaCl, and BHT plus NaCl were statistically significant. NaCl given alone after MNNG administration also significantly increased the incidence of papillomas in the forestomach. Incidences of glandular stomach tumors, adenomas and carcinomas were not affected by any of the subsequent treatments. No tumors of the stomach developed in the groups given BHA, BHT, and NaCl without MNNG pretreatment. Thus the present experiment revealed that BHA and NaCl but not BHT exert promoting activity on MNNG-induced forestomach carcinogenesis in rats and that, when BHA and BHT were given with NaCl, promotion was more marked, suggesting a synergistic effect on tumor promotion.     

High susceptibility of analbuminemic rats to gastric tumor induction by N-methyl-N'-nitro-N-nitrosoguanidine

Analbuminemic rats were found to be highly susceptible to induction of gastric tumors by N-methyl-N'-nitro-N-nitrosoguanidine (67-83 micrograms/ml) given to the rats in drinking water for 32 weeks. The rats were sacrificed at experimental week 44. Gastric tumors were found in 12 of 17 analbuminemic rats (70%) and in 8 of 21 normal rats (38%). Intestinal tumors developed in 7 of 17 (41%) analbuminemic rats and in 9 of 21 (42%) normal rats.     

Effects of phenolic antioxidants in low dose combination on forestomach carcinogenesis in rats pretreated with N-methyl-N'-nitro-N-nitrosoguanidine

The combined effects of low doses of promoters or carcinogens on two-stage forestomach carcinogenesis were examined in rats pretreated with N-methyl-N'-nitro-N-nitrosoguanidine. Groups of 15 rats were given a single 150 mg/kg body weight intragastric dose of N-methyl-N'-nitro-N-nitrosoguanidine. Starting 1 week later they were fed a diet containing low doses of known forestomach promoters/carcinogens (0.5% caffeic acid, 0.2% catechol, 0.5% butylated hydroxyanisole, or 0.25% 2-tert-butyl-4-methylphenol), alone or in combination, or basal diet without antioxidant supplement for 35 weeks. Histopathological examination revealed the incidences of forestomach squamous cell carcinomas in animals treated with N-methyl-N'-nitro-N-nitrosoguanidine followed by caffeic acid, catechol, butylated hydroxyanisole, 2-tert-butyl-4-methylphenol, and basal diet to be 27, 20, 13, 13, and 7%, respectively, whereas the incidence increased to 80% by the combined treatment with these four chemicals. The present results thus show that although the low doses of individual promoters/carcinogens did not have significant promoting activity, their combination exerted a strong enhancing influence on rat forestomach carcinogenesis. The findings indicate the importance of summation and synergism at a low dose for agents present in the human environment.     

Effects of bilateral and unilateral vagotomy on gastric carcinogenesis induced by N-methyl-N'-nitro-N-nitrosoguanidine in Wistar rats

After administration of N-methyl-N'-nitro-N-nitrosoguanidine for 15 weeks, the effects of bilateral, anterior and posterior vagotomy on the incidence, number and location of gastric adenocarcinomas, gastric acid secretion and cell proliferation of the gastric mucosa were investigated in inbred Wistar rats. Bilateral or anterior vagotomy, but not posterior vagotomy, significantly increased the incidence and number of adenocarcinomas at experimental week 52. In sham-operated control rats and rats subjected to bilateral vagotomy, there was no significant difference between the incidence or number of gastric tumors in the anterior and posterior walls. After anterior and posterior vagal denervation, however, there were significantly more gastric cancers on the denervated side than on the other. Bilateral and unilateral vagotomy resulted in significantly reduced gastric acid secretion by experimental weeks 25 and 52. Bilateral vagotomy significantly increased the labelling indices of both the fundic and antral mucosa at both times, but did not cause any significant difference between those of the anterior and posterior wall. Anterior or posterior vagotomy resulted in a significant increase in the labelling indices of both the fundic and antral mucosa on the denervated side. These findings indicate that the vagal nerve exerts a trophic action on the gastric mucosa, and that the promoting effect of vagotomy on gastric carcinogenesis may be related to its effect in increasing proliferation of cells in the antral mucosa.     

Enhancing effects of N-ethyl-N'-nitro-N-nitrosoguanidine and sodium taurocholate on development of pepsinogen 1 decreased pyloric glands in rats initiated with N-methyl-N'-nitro-N-nitrosoguanidine

Sequential quantitative analyses were made of pepsinogen 1 (Pg 1) decreased pyloric glands after treating male WKY rats first with N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) and then with N-ethyl-N'-nitro-N-nitrosoguanidine (ENNG) as a second gastric carcinogen or sodium taurocholate (Na-TC) as a gastric promoter. Animals received a single dose of MNNG (160 mg/kg body weight) by gastric intubation followed two weeks later by either ENNG in drinking water (100 micrograms/ml) (group 1), basal diet containing 0.25% Na-TC (group 2), or basal diet and tap water (group 3), from weeks 3 to 24. Animals were sacrificed at weeks 8, 12, 16, 20 and 24. Sections of the pyloric mucosa were investigated for Pg 1 immunostaining. In comparison with group 3, induction of Pg 1 decreased pyloric glands was significantly enhanced by ENNG from week 8 and by Na-TC from week 16. The former exerted a significantly stronger effect at each time point. The results suggest that Pg 1 decreased pyloric glands represent a good marker for early detection of gastric carcinogens and promoters in in vivo test systems.     

Effects of propranolol and cimetidine on cysteamine inhibition of gastric carcinogenesis induced in Wistar rats by N-methyl-N'-nitro-N-nitrosoguanidine

The effects of propranolol and cimetidine on inhibition by cysteamine (2-aminoethanethiol hydrochloride) of gastric carcinogenesis induced by N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) and on gastric acid secretion, serum gastrin level, and labelling index of the gastric mucosa were investigated in inbred Wistar rats. Rats received alternate-day injections of cysteamine (25 mg/kg body weight) with or without propranolol (dl-propranolol hydrochloride) (2 mg/kg bw) or cimetidine (50 mg/kg bw) in depot form after 25 weeks of oral treatment with MNNG. Prolonged administration of cysteamine significantly reduced the incidence of adenocarcinoma of the glandular stomach. A combination of cysteamine and propranolol significantly accelerated the inhibitory effect of cysteamine on gastric carcinogenesis. However, with concomitant administration of cysteamine and cimetidine, the incidence of adenocarcinoma was slightly but not significantly increased as compared to that after treatment with cysteamine alone. Administration of cysteamine caused a significant increase in gastric acid secretion and serum gastrin level, and a significant decrease in the labelling index of the antral mucosa. A combination of cysteamine and propranolol significantly increased gastric acid secretion by cysteamine alone and significantly decreased the labelling index of the antral mucosa. With this treatment, the serum gastrin level was significantly higher than the basal level, but the stimulated serum gastrin level was significantly lower than observed that after administration of cysteamine alone. In contrast, concomitant administration of cysteamine and cimetidine caused a significant decrease in gastric acid secretion and significant increase in the serum gastrin level as compared to the levels seen after treatment with cysteamine alone, but had no influence on the labelling index of the antral mucosa. These findings indicate that hypersecretion of acid, but not hypergastrinemia associated with hyposecretion of acid or achlorhydria, exerts a protective effect against gastric carcinogenesis, and that this effect may be related to its activity in decreasing proliferation of the antral mucosa.     

Effects of sodium L-ascorbate and related compounds on rat stomach carcinogenesis initiated by N-methyl-N'-nitro-N-nitrosoguanidine

The modifying effects of 3 antioxidants, sodium L-ascorbate (SA), ascorbic acid (AA) and sodium erythorbate (SE) on two-stage gastric carcinogenesis in F344 rats initiated with N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) were investigated. Administration of 5% SE in the diet significantly decreased the incidence of dysplasia of the pylorus and, more marginally the incidence of papilloma of the forestomach, whereas administration of 5% and 1% SA and 5% AA in the diet was not associated with effect. These results suggest that SE exerts a weak inhibitory effect on gastric carcinogenesis.     

Promoting effect of intestinal Pseudomonas aeruginosa on gastric tumorigenesis in rats with N-methyl-N'-nitro-N-nitrosoguanidine

The effect of Pseudomonas aeruginosa colonizing in the gut on the development of gastrointestinal tumors was studied in rats treated orally with N-methyl-N'-nitro-N-nitrosoguanidine (MNNG). The animals were inoculated with or accidentally colonized by P. aeruginosa after MNNG treatment. In 3 serial experiments, P. aeruginosa-positive, MNNG-treated rats consistently showed a significantly higher incidence of gastric tumors than P. aeruginosa-negative, MNNG-treated animals. The total incidence of gastric tumors was 40% (34/86) in the former, and only 11% (7/61) in the latter. The development of tumors in the small intestine was not likely to be influenced by P. aeruginosa colonization. It is concluded that P. aeruginosa in the gut plays a promoting role in gastric tumorigenesis in rats orally administered with MNNG.     

Promotion by bombesin of gastric carcinogenesis induced by N-methyl-N'-nitro-N-nitrosoguanidine in Wistar rats

The effects of bombesin on the incidence, number, histological type, and depth of involvement of gastric cancers induced by N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) were investigated in male Wistar rats. Rats received alternate-day s.c. administration of 20 or 40 micrograms/kg body weight of bombesin in depot form after p.o. treatment with the carcinogen for 25 weeks. Prolonged administration of bombesin at 40 micrograms/kg led to a significant increase in the incidence and number per rat of gastric cancers of the glandular stomach at Week 52. In rats that had received alternate-day injections of 20 micrograms/kg of bombesin, the number of gastric cancers per rat, but not the incidence of cancer, was significantly more than in untreated rats. However, bombesin at both dosages did not affect the histological appearance of the lesions or their depth of involvement. At Weeks 30 and 52, norepinephrine concentrations in the fundic and antral portion of the gastric walls and labeling indices in the antral and fundic mucosae were significantly higher in rats treated with bombesin at both dosages than in untreated rats. These findings indicate that bombesin enhances gastric carcinogenesis after administration of N-methyl-N'-nitro-N-nitrosoguanidine is stopped and that this effect may be related to its effects in increasing tissue norepinephrine concentrations in the stomach wall and increasing cell proliferation in the gastric mucosa.     

Promotion by ethanol of gastric carcinogenesis induced by N-methyl-N'-nitro-N-nitrosoguanidine in Wistar rats

The effects of ethanol (EtOH) on the incidence and histology of gastric cancers induced by N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) were investigated in Wistar rats. The animals received alternate-day i.p. injections of 2.5 ml kg-1 body weight of 20% EtOH in 0.9% NaCl solution after 20 weeks of oral treatment with MNNG. Prolonged administration of EtOH resulted in a significant increase in the incidence and number of gastric cancers of the glandular stomach in week 52. However, it had no influence on the histological types of the gastric cancers. Furthermore, it caused a significant increase in the labelling index of the epithelial cells of the antrum in week 52. These findings indicate that EtOH promotes gastric carcinogenesis, and that this effect may be related to its effect in increasing proliferation of the antral epithelial cells.     

Promotion by neurotensin of gastric carcinogenesis induced by N-methyl-N'-nitro-N-nitrosoguanidine in Wistar rats

The effects of neurotensin on the incidence and histology of gastric cancers induced by N-methyl-N'-nitro-N-nitrosoguanidine were investigated in Wistar rats. Rats were given 100 or 200 micrograms per kg of body weight of neurotensin s.c. every other day in depot form after 25 wk of p.o. treatment with the carcinogen. Prolonged alternate-day administration of neurotensin at 200 micrograms per kg of body weight resulted in a significant increase in the incidence of gastric cancers of the glandular stomach by Wk 52. However, it did not influence the histological appearance of the gastric cancers. Furthermore, it caused a significant increase in the labeling indices of the epithelial cells of the antrum and of gastric cancers. In contrast, the administration of neurotensin at 100 micrograms per kg of body weight had a slight, but not significant, influence on the development of gastric cancers. These findings indicate that neurotensin promotes gastric carcinogenesis, and that this effect may be related to its effect in increasing proliferation of epithelial cells in the antral mucosa and in gastric cancers.     

Promotion by nialamide of gastric carcinogenesis induced by N-methyl-N'-nitro-N-nitrosoguanidine in Wistar rats

The effects of nialamide, a monoamine oxidase inhibitor, on the incidence, number, and histology of gastric cancers induced by N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) were investigated in male Wistar rats. Rats were given subcutaneously 50 mg/kg body weight of nialamide in depot form every other day after 25 weeks of oral treatment with MNNG. Prolonged alternate-day administration of nialamide caused a significant increase in the incidence and number of gastric cancers of the glandular stomach in week 52. However, it did not affect the histology of the cancers. Nialamide also caused a significant increase in tissue norepinephrine concentrations in the gastric wall and in the labeling indices of the gastric mucosae. However, nialamide had no influence on serum gastrin levels in the fasting state and after re-feeding. These findings indicate that nialamide promotes gastric carcinogenesis and that this may be related to its effects in increasing norepinephrine in the gastric wall and stimulating proliferation of gastric epithelial cells.     

Enhancement by aminoguanidine of gastric carcinogenesis induced by N-methyl-N'-nitro-N-nitrosoguanidine in Wistar rats

The effects of prolonged administration of aminoguanidine on gastric carcinogenesis induced by N-methyl-N-nitro-N-nitrosoguanidine (MNNG), the ornithine decarboxylase activity of the gastric wall, and the labeling index of the gastric mucosa were investigated in inbred Wistar rats. The rats received 12.5 or 25.0 mg/kg body weight of aminoguanidine s.c. every other day after oral MNNG treatment for 25 weeks. Prolonged administration of aminoguanidine at 25.0 mg/kg body weight, but not at 12.5 mg/kg body weight, significantly increased the incidence of gastric cancers in experimental week 52. However, it did not influence the histologic types of gastric cancers. Aminoguanidine at 25.0 mg/kg body weight also significantly increased the ornithine decarboxylase activity of the antral portion of the gastric wall and the labeling index of the antral epithelial cells. These findings indicate that aminoguanidine enhances gastric carcinogenesis and suggest that its effect may be related to increased proliferation of antral epithelial cells.     

Promotion by vagotomy of gastric carcinogenesis induced by N-methyl-N'-nitro-N-nitrosoguanidine in Wistar rats

The effects of truncal vagotomy after administration of N-methyl-N'-nitro-N-nitrosoguanidine on the incidence and number of gastric carcinomas and gastric acid secretion, gastrin secretion, antral pH, and duodenal reflux were investigated in inbred Wistar rats. Rats were subjected to truncal vagotomy after N-methyl-N'-nitro-N-nitrosoguanidine treatment. Vagotomy significantly increased the incidence and number of adenocarcinomas of the glandular stomach. It also resulted in significantly more atypical glandular hyperplasias, which are precursors of gastric cancer. Furthermore, it caused a decrease in gastric secretion and an increase in mucosal pH in the antrum but did not increase duodenal reflux. These findings indicate that vagotomy has a promoting effect on the development of gastric cancers. The reduced gastric acid secretion, but not duodenal reflux, may be related to this increased incidence of gastric cancer.     

Enhancement of experimental gastric carcinogenesis induced in spontaneously hypertensive rats by N-methyl-N'-nitro-N-nitrosoguanidine

The incidence, number, and histological types of gastric cancers induced by N-methyl-N'-nitro-N-nitrosoguanidine and the tissue norepinephrine concentration of the gastric wall were investigated in spontaneously hypertensive rats and in control Wistar Kyoto rats and Wistar rats. All rats were given drinking water containing 25 micrograms/ml of N-methyl-N'-nitro-N-nitrosoguanidine for 25 weeks. During Week 52, the incidence and number per rat of gastric cancers were significantly greater in spontaneously hypertensive rats than in Wistar Kyoto and Wistar rats. All tumors induced in the glandular stomach were adenocarcinomas, but no significant difference was found in the histological types of adenocarcinoma in the three strains of rats. At Weeks 15, 30, and 52, norepinephrine concentrations in the fundic and antral portion of the gastric walls and labeling indices in the antral and fundic mucosa were significantly higher in spontaneously hypertensive rats than in Wistar Kyoto and/or Wistar rats. These findings indicate that increased sympathetic nervous system activity enhances the development of gastric cancers, but immunological dysfunction in spontaneously hypertensive rats may contribute to the increased susceptibility to gastric cancer.     

Effects of ethanol, potassium metabisulfite, formaldehyde and hydrogen peroxide on gastric carcinogenesis in rats after initiation with N-methyl-N'-nitro-N-nitrosoguanidine

Ethanol, potassium metabisulfite, formaldehyde and hydrogen peroxide were tested for tumor-promoting activity in a two-stage stomach carcinogenesis experiment. Male outbred Wistar rats were given N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) in the drinking water (100 mg/liter) and a diet supplemented with 10% sodium chloride for 8 weeks. Thereafter, they were maintained on drinking water containing either 10% ethanol, 1% potassium metabisulfite, 0.5% formalin (formaldehyde) or 1% hydrogen peroxide for 32 weeks and then sacrificed for necropsy and histological examination. In the pylorus of the glandular stomach, potassium metabisulfite and formaldehyde significantly increased the incidence of adenocarcinoma after initiation with MNNG and sodium chloride. Hydrogen peroxide did not enhance the tumor yield, and ethanol showed a tendency to decrease neoplastic development. In the forestomach the incidence of squamous cell papilloma was significantly increased in the groups given hydrogen peroxide or formaldehyde, irrespective of prior initiation. Duodenal adenocarcinoma was induced by the initiation alone (10%) and the incidence was not affected by the subsequent treatments. The results indicate that potassium metabisulfite and formaldehyde both exert tumor-promoting activity in the rat glandular stomach.     

Enhanced induction by high-cholesterol diet of remnant gastric carcinogenesis by N-methyl-N'-nitro-N-nitrosoguanidine in rats

The influence of bile acids on the development of remnant gastric carcinoma was examined by investigating the incidence of carcinogenesis in noninbred male Wistar rats treated orally with N-methyl-N'-nitro-N-nitrosoguanidine (MNNG; CAS 70-25-7; 1-methyl-3-nitro-1-nitrosoguanidine) and fed a diet containing 1% cholesterol. The high-cholesterol diet did not influence the incidence of carcinoma in the nongastrectomized, MNNG-treated groups of rats. However, in the gastrectomized groups, the incidence of carcinoma was significantly higher in the group given the high-cholesterol diet (60.6%) than in the group given a normal diet (35.5%). Histologically undifferentiated adenocarcinoma was recognized more frequently in the high-cholesterol-diet group. Three gastrectomized rats not treated with MNNG but fed the high-cholesterol diet developed remnant gastric carcinoma (13%), whereas none of the rats given the normal diet did. Because the fecal excretion of bile acids increased significantly in the rats fed the high-cholesterol diet and the gastroduodenal reflux of bile acids was probably accelerated, the increase in the incidence of carcinogenesis in the remnant stomach was considered to be the result of the increase in the reflux of bile acids evoked by a high-cholesterol diet.     

Inhibition by amiloride of gastric carcinogenesis induced by N-methyl-N'-nitro-N-nitrosoguanidine in Wistar rats.

The effects of amiloride on the incidence and histological types of gastric cancers in Wistar rats induced by N-methyl-N''-nitro-N-nitrosoguanidine (MNNG), and on the labelling index and proliferative fraction of gastric mucosa were investigated. After oral treatment with MNNG for 25 weeks, rats received s.c. injections of amiloride (0.25 mg kg-1 or 5.0 mg kg-1 body weight) in depot form every other day until the end of the experiment. Prolonged administration of 5.0 mg kg-1, but not 2.5 mg kg-1 of amiloride significantly decreased the incidence of gastric cancers in Week 52. However, it did not influence the histological features of the gastric cancers. It also significantly decreased the labelling index and proliferative fraction of the antral mucosa. These findings indicate that amiloride inhibits the development of gastric cancers, and that its effect may be related to its effect in decreasing cell proliferation of the antral mucosa.     

Enhancement by thyroxine of gastric carcinogenesis induced by N-methyl-N'-nitro-N-nitrosoguanidine in Wistar rats.

The affects of L-thyroxine (T4) on the incidence and histology of gastric cancers induced by N-methyl-N''-nitro-N-nitrosoguanidine (MNNG), and on the labelling index of gastric mucosal epithelial cells were investigated in Wistar rats. After oral treatment with MNNG for 25 weeks, the rats received s.c. injections of T4 (0.2 microgram kg-1) in depot form every other day until the end of the experiment in Week 52. This long-term treatment with T4 significantly increased the incidence of gastric cancers in Week 52. However, it did not influence the histological type of the gastric cancers. It also caused significant increases in the labelling indices of the fundic and antral epithelial cells. These findings indicate that T4 enhances the development of gastric cancers, and that its effect may be related to its effect in increasing proliferation of gastric epithelial cells.