Synergism between Sodium Chloride and Sodium Taurocholate and Development of Pepsinogen-altered Pyloric Glands: Relevance to a Medium-term Bioassay System for Gastric Carcinogens and Promoters in Rats

In an approach to early detection of gastric carcinogens and promoters in an in vivo test system, promotion by sodium chloride (NaCl) and the synergistic effects of NaCl and sodium taurocholate (Na-TC) on development of pepsinogen-altered pyloric glands (PAPG) in rat glandular stomach after initiation with N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) were investigated. A total of 205 male WKY/NCrj rats were divided into 8 groups. Group 1 was given a single dose of MNNG of 160 mg/ kg body weight by gastric intubation, and starting 2 weeks later basal diet containing Na-TC for 18 weeks. In addition, 1 ml doses of saturated NaCl solution were given by gastric intubation at weeks 4, 6, 8 and 10. Similarly, group 2 was treated with MNNG and Na-TC, while group 3 animals received MNNG and NaCl. Group 4 was given MNNG alone. Groups 5–8 served as equivalent controls without MNNG initiation. The results revealed significantly enhanced induction of immunohisto-chemically defined PAPG in the Na-TC + NaCl ( P < 0.001), Na-TC ( P <0.01) and NaCl ( P <0.01) treated animals initiated with MNNG. Sodium chloride demonstrated a clear synergistic effect with Na-TC in promoting the development of PAPG, suggesting possible advantage for its use in medium-term in vivo assays for detection of gastric carcinogens and promoters.     

Synergism between sodium chloride and sodium taurocholate and development of pepsinogen-altered pyloric glands: relevance to a medium-term bioassay system for gastric carcinogens and promoters in rats

In an approach to early detection of gastric carcinogens and promoters in an in vivo test system, promotion by sodium chloride (NaCl) and the synergistic effects of NaCl and sodium taurocholate (Na-TC) on development of pepsinogen-altered pyloric glands (PAPG) in rat glandular stomach after initiation with N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) were investigated. A total of 205 male WKY/NCrj rats were divided into 8 groups. Group 1 was given a single dose of MNNG of 160 mg/kg body weight by gastric intubation, and starting 2 weeks later basal diet containing Na-TC for 18 weeks. In addition, 1 ml doses of saturated NaCl solution were given by gastric intubation at weeks 4, 6, 8 and 10. Similarly, group 2 was treated with MNNG and Na-TC, while group 3 animals received MNNG and NaCl. Group 4 was given MNNG alone. Groups 5-8 served as equivalent controls without MNNG initiation. The results revealed significantly enhanced induction of immunohistochemically defined PAPG in the Na-TC + NaCl (P less than 0.001), Na-TC (P less than 0.01) and NaCl (P less than 0.01) treated animals initiated with MNNG. Sodium chloride demonstrated a clear synergistic effect with Na-TC in promoting the development of PAPG, suggesting possible advantage for its use in medium-term in vivo assays for detection of gastric carcinogens and promoters.     

Immunohistochemical demonstration of induction of pyloric glands with low pepsinogen 1 (Pg 1) content in rat stomach by N-methyl-N'-nitro-N-nitrosoguanidine

Three groups of male Fischer rats were given single doses of N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) at 160 mg (group 1), 80 mg (group 2) and 40 mg (group 3)/kg body weight by gastric intubation. A fourth group was given drinking water containing 100 micrograms/ml of MNNG for 2 weeks, and a fifth group served as a control. Rats were killed in weeks 5, 8 and 12. Serial sections of the pyloric mucosa were examined by paradoxical concanavalin A (Con A) staining and pepsinogen isozyme 1 (Pg 1) immunostaining. All pyloric glands contained class III mucin as detected by paradoxical Con A staining. Most pyloric glands had a high Pg 1 content, but a few stained only weakly if at all. The percentage and number (No./500 normal-looking pyloric glands) of pyloric glands with a low Pg 1 content were 50.0 and 0.2 +/- 0.4 (week 5), 87.5 and 0.5 +/- 0.4 (week 8) and 100.0 and 1.2 +/- 1.0 (week 12) in group 1, 50.0 and 0.2 +/- 0.3 (week 8) and 87.5 and 0.5 +/- 0.4 (week 12) in group 2, and 30.0 and 0.2 +/- 0.4 (week 12) in group 4. No pyloric glands with a low Pg 1 content were found in groups 3 and 5. Thus the results showed significant dose-dependent induction (P less than 0.05-0.01) of altered pyloric glands demonstrating reduced Pg 1 content and their earlier appearance in groups given higher doses of MNNG. The results suggest that the appearance of pyloric glands with a low Pg 1 content may be a preneoplastic change in gastric carcinogenesis.     

Effects of bile acids on development of pepsinogen-altered pyloric glands in rats

The effects of dietary bile acids on the development of pepsinogenaltered pyloric glands (PAPG) were examined. Male WKY/NCrj rats were given a single dose of 160 mg/kg body wt. of N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) by gastric intubation and fed basal diet containing 0.3% sodium taurocholate (Na-TC), 0.3% sodium cholate (Na-C), 0.3% sodium glycocholate (Na-GC), 0.3% sodium tauroglycocholate, 0.3% sodium deoxycholate, 0.1% chenodeoxycholic acid or 0.5% lithocholic acid for 14 weeks. All rats also received 1 ml of saturated NaCl solution 4 times by i.g. intubation. At the end of week 16, the animals were killed, and the number of PAPG per cm of mucosal length was determined immunohistochemically. Na-TC, Na-C and Na-GC significantly increased the number of PAPG over the control value, suggesting that they may have activity to promote gastric carcinogenesis.     

Immunohistochemical demonstration of pyloric gland-type cells with low-pepsinogen isozyme 1 in preneoplastic and neoplastic tissues of rat stomachs treated with N-methyl-N'-nitro-N-nitrosoguanidine

The appearance of pyloric gland-type cells with a low pepsinogen isozyme 1 (Pg 1) content in the stomach mucosa of F344/Du rats during stomach carcinogenesis was examined by a combination of paradoxical concanavalin A (Con A) staining and immunohistochemical staining for Pg 1. Male F344 rats were given drinking water containing 100 micrograms N-methyl-N'-nitro-N-nitrosoguanidine [(MNNG) CAS: 70-25-7]/ml for 30 weeks and then normal tap water and were killed in week 10, 20, 30, 40, 50, or 70. Untreated rats were killed in week 30 or 70. Serial sections of pyloric mucosa were stained by paradoxical Con A staining and Pg 1 immunostaining. After MNNG treatment, tissues showing changes were classified into normal-looking pyloric mucosa with a low Pg 1 content, mucosa showing atrophic or hyperplastic changes, adenomatous hyperplasia, and adenocarcinoma. From the results of paradoxical Con A staining and Pg 1 immunostaining, the cells in lesions were classified into gastric types (surface mucous cell type and pyloric gland cell type) and intestinal types (intestinal-absorptive cell type and goblet cell type). In this experiment, the cells in lesions were mainly of the gastric cell types. All pyloric glands of control rats in weeks 30 and 70 contained class III mucins and had a high Pg 1 content demonstrated immunohistochemically. After MNNG treatment, class III mucin-positive pyloric glands with a low Pg 1 content in normal-looking pyloric mucosa were found from week 10; subsequently, their number increased with time. Changed mucosa was found from week 20, and the area of cells of the pyloric gland cell type with little or no Pg 1 in changed mucosa was about 30% of the area of cells of the pyloric gland cell type. Adenomatous hyperplasias were found from week 30; adenocarcinomas were found from week 50. Almost all cells of the pyloric gland cell type (greater than 95%) in areas of adenomatous hyperplasia and adenocarcinomas had little or no Pg 1 content. The present results suggested that the appearance of pyloric glands with a low Pg 1 content in normal-looking mucosa might be an immunohistochemically detectable preneoplastic change preceding morphologically detectable preneoplastic changes in stomach carcinogenesis.     

Dose-dependent Induction of Both Pepsinogen-altered Pyloric Glands and Adenocarcinomas in the Glandular Stomach of C3H Mice Treated with N-Methyl-N-nitrosourea

The dose-response relation for the appearance of pepsinogen isozyme 1 (Pg l)-altered pyloric glands (PAPG) and the related induction of adenocarcinomas were examined in male C3H mice given N -methyl- N -nitrosourea (MNU) in their drinking water at the concentration of 120 ppm (group 1), 60 ppm (group 2), 30 ppm (group 3) or 0 ppm (group 4) for 30 weeks and then normal tap water. Animals were killed at weeks 10, 30 and 42. Adenomatous hyperplasias and adenocarcinomas were noted from week 30 and their induction was dose-dependent at week 42. Almost all cells of pyloric gland cell type in those lesions had little or no immunohistochemically demonstratable Pg 1 content, as was also the case for the cells in PAPG, whose numbers per 100 normal-appearing pyloric glands were found to be MNU dose-dependent at all experimental time points. The numbers of PAPG at week 10 significantly correlated with the incidences of adenomatous hyperplasias and adenocarcinomas at week 42. Investigation of proliferation by immunohistochemical detection of bromodeoxyuridine (BrdU) labeling in the PAPG at week 10 demonstrated elevation (P <0.05) as compared to normal pyloric glands. Intestinal metaplasia was not a feature in the present experiment and the results suggest that in mice, PAPG might be a preneoplastic lesion involved in gastric chemical carcinogenesis.     

Effects of NaCl, Tween 60 and a low dose of N-ethyl-N' -nitro-N-nitrosoguanidine on gastric carcinogenesis of rat given a single dose of N-methyl-N' -nitro-N-nitrosoguanidine

Effects of NaCI, Tween 60 and N-ethyl-N' -nitro-N-nitrosoguanidine(ENNG) on gastric carcinogenesis were investigated in male Wistarrats. Animals received a single dose of N-methyl-N' -nitro-N-nitrosoguanidlne(MNNG) at 250 mg/ kg body weight by gastric tube followed oneweek later by either 10% NaCI in their diet, twice-weekly applicationsof 1 ml of saturated NaCI solution by gastric tube, 1.0% Tween60 in their drinking water or 0.0005% ENNG in their drinkingwater. One group of rats were given MNNG 24 h after a singleapplication of 1 ml of saturated NaCI solution to investigatethe effect of NaCI on initiation. A single dose of MNNG to ratsresulted in development of multiple epithelial tumors in theforestomach and no epithelial tumors in the glandular stomachafter 52 weeks. There were no differences in tumor incidencesof the forestomach and glandular stomach between experimentalgroups which were given a subsequent treatment with NaCI orTween 60 and the control group with MNNG alone. ENNG significantlyenhanced the tumor induction in the glandular stomach, whileENNG alone did not induce any tumors in the stomach. The NaCItreatment prior to MNNG administration also increased tumordevelopment in the glandular stomach but not in the forestomach.     

Proliferation kinetics of pepsinogen altered pyloric gland cells in rats treated with N-methyl-N'-nitro-N-nitrosoguanidine

The cell kinetics of pepsinogen isozyme 1 altered pyloric gland (PAPG) cells with low pepsinogen isozyme 1 (Pg 1) content were analysed using double immunohistochemical staining for bromodeoxyuridine (BrdU) incorporation and Pg 1 in male WKY/NCrj rats treated with N-methyl-N'-nitro-N-nitrosoguanidine (MNNG). After administration of 100 micrograms/ml MNNG for 10 weeks in the drinking water, carcinogenic insult was terminated and the animals killed two weeks later. BrdU was given either as a single i.p. injection (100 mg/kg b.w.) 1 h prior to death or continuously by osmotic minipump (120 micrograms/h) for 4, 7 and 10 days before killing. Immunogold-silver staining was used to detect BrdU and the avidin-biotin-peroxidase complex method adopted for demonstration of Pg 1. PAPG were found only in the MNNG treated group: their frequency was 4.1 +/- 0.6 per 100 pyloric glands. Almost no normal pyloric gland cells with high Pg 1 content demonstrated incorporation after BrdU flash labelling. However, a few pyloric gland cells in PAPG were labelled. The number of labelled cells in the pyloric columns containing PAPG was larger (P less than 0.05) than in normal pyloric columns. After continuous BrdU administration, the life span of cells comprising PAPG was estimated to be approximately 6-8 days while that of normal pyloric gland cells was approximately 11-13 days. Thus, the data indicate that PAPG cells demonstrate a degree of independence from surrounding pyloric glands with regard to proliferation kinetics, suggesting that PAPG is a preneoplastic lesion involved in gastric carcinogenesis.     

DNA mutilation of the pepsin 1 gene during rat glandular stomach carcinogensis induced by N-methyl-N'-nitro-N-nitrosoguanidine or catechol

The methylation patterns of the rat pepsinogen 1 (Pg1) genein preneoplastic and neoplastic stomach lesions induced by genotoxicN-methyl-N'-nitro-N-nitrosoguanidine (MNNG) or the non-genotoxiccarcinogen catechol were investigated. Male WKY/Ncrj rats weregiven MNNG in their drinking water (50 mg/l) for 30 weeks or0.8% catechol throughout the experiment (60 weeks). MNNG inducedPg1 altered pyloric glands (PAPG), adenomatous hyperplasiasand well-differentiated adenocarcinomas. Catechol also inducedPAPG and adenomatous hyperplasias although cancers did not develop.Adenomatous hyperplasias and adenocarcinomas all consideredof gastric type cells resembling surface mucous cells or pyloricgland cells with little or no Pg1 expression. In MNNG-inducedstomach cancers generally lacking Pg1, altered Pg1 gene methylationwas observed with both CCGG and GCGC sites being methylatedmore than normal pyloric mucosa. MNNG or catechol-induced adenomatoushyperplasias also demonstrated essentially the same methylationchanges in the CCGG, but not in the GCGC sites. In the mucosacontaining PAPG in groups treated with MNNG or catechol themethylation patterns of the Pg1 gene were quite similar to thoseof normal pyloric mucosa, although the CCGG sites tended todemonstrate slightly increased methylation. The results suggestthat the altered methylation of the Pg1 gene observed in stomachcancers is acquired early in the carcinogenic process and progressivemethylation changes occur with tumor development.     

Changes in pepsinogen isozymes in stomach carcinogenesis induced in rats by N-methyl-N'-nitro-N-nitrosoguanidine.

Changes in the isozymes of pepsinogen (Pg) separated from the glandular stomachs of rats were studied by polyacrylamide gel electrophoresis during carcinogenesis induced by N-methyl-N'-nitro-N-nitrosoguanidine (MNNG), from the beginning of MNNG administration to 3 months after the end of its 7-month regimen. In 13 of 25 rats killed successively, one (Pg 1) of the three pepsinogen isozymes (Pg 1, 3, 4) normally present in the pyloric mucosa had decreased or disappeared. It decreas was observed from 1 week after the beginning of MNNG treatment to at least 3 months after the end of the 7-month MNNG administration. Remarkable histopathologic changes were found from 8 months after MNNG was given, and rats showing such unusual histopathologic alterations also had changes in their pepsinogen isozyme pattern. In 4 of 27 rats, two (Pg 1, 2) of the four isozymes of pepsinogen (Pg 1-4) in the fundic mucosa decreased or disappeared from 3 months after the beginning of MNNG treatment to at least 2 months after the end of its 7-month administration. Histopathologic changes induced by MNNG were not as remarkable in the fundic mucosa as in the pyloric mucosa.     

Effects of 4-week treatment with gastric carcinogens and enhancing agents on proliferation of gastric mucosa cells in rats

Catechol and N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) are gastric carcinogens in rats. Catechol, sodium chloride and bile salts have enhancing effects on gastric carcinogenesis induced by MNNG in rats. The effects of these compounds on proliferation of pyloric mucosa cells in male F344 rats were examined immunohistochemically using bromodeoxyuridine (BrdU) and anti-BrdU monoclonal antibody. Rats were given MNNG (83 micrograms/ml in their drinking water), catechol (0.8% in their diet), sodium taurocholate (0.3% in their diet), sodium taurodeoxycholate (0.3% in their diet), or sodium chloride (10% in their diet or by intragastric administration of 1 ml of saturated solution once a week) for 4 weeks. All these treatments markedly enhanced cell proliferation of the pyloric epithelium, suggesting the importance of enhanced cell proliferation in the development of gastric cancer.     

Effects of butylated hydroxyanisole pretreatment on low dose N-methyl-N'-nitro-N-nitrosoguanidine- or N,N-dibutylnitrosamine-induced rat forestomach or esophageal carcinogenesis

The effects of butylated hydroxyanisole (BHA) pretreatment on subsequent low dose N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) or N,N-dibutylnitrosamine (DBN) treatment on forestomach or esophageal carcinogenesis were investigated in male F344 rats. Groups of animals were pretreated with 2% BHA or basal diet alone for 24 weeks and then were given 20 mg/kg body wt MNNG once every 2 weeks, 0.025% DBN in drinking water continuously or basal diet alone for the subsequent 24 weeks. Further groups of rats were similarly treated with BHA or basal diet alone for 24 weeks, placed on basal diet for the next 24 weeks and then treated with MNNG, DBN or basal diet alone for the subsequent 24 weeks. Animals were killed 48 or 72 weeks after the beginning of the experiment. Histopathological examination showed that the incidence of forestomach tumors was not significantly affected by the BHA pretreatment in the MNNG-treated groups. On the other hand, the incidence of esophageal squamous cell carcinomas was lower in the group pretreated with BHA followed by DBN than in that treated with basal diet followed by DBN (48 week experiment). There was no significant difference in esophageal tumor incidence in the 72 week experiment. The results thus indicate that continuous treatment with 2% BHA for 24 weeks does not exert initiating activity on forestomach and esophageal epithelia.     

Enhancing effects of various gastric carcinogens on development of pepsinogen-altered pyloric glands in rats

To assess the possibility of establishing an in vivo, medium-term bioassay system for gastric carcinogens and promoters, a total of 220 male WKY rats were divided into two groups. Group 1 animals were treated first with a single dose of N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) (160 mg/kg body wt) and starting 2 weeks later administrated one of five gastric carcinogens, a gastric promoter, one of five non-gastric carcinogens or no treatment, as a control, for 14 weeks. Saturated sodium chloride solution (1 ml) treatments were given by gastric intubation at weeks 4, 6, 8 and 10. Group 2 rats received 1 ml of DMSO instead of MNNG and were then treated in the same way as group 1. Analysis of pyloric mucosa sections for pepsinogen altered pyloric glands (PAPG) detected immunohistochemically after the animals were killed at week 16 revealed increased lesion numbers in group 1, with all gastric carcinogens and promoters examined. However, none of the five non-gastric carcinogens exerted any significant modification of PAPG development. The results strongly suggest that the experimental protocol consisting of the following four components: (i) adoption of PAPG as the end-point marker lesion; (ii) single dose of MNNG as initiator; (iii) test chemical administration for 14 weeks; and (iv) administration of saturated sodium chloride solution during the test chemical exposure, could be used effectively for the detection of gastric carcinogens as well as promoters of gastric carcinogenesis in a relatively short time period.     

Inhibition of N-ethyl-N'-nitro-N-nitrosoguanidine-induced Duodenal Tumorigenesis in Mice by . Whole-leaf Aloe arborescens Miller var. natalensis Berger

We examined the modifying effects of freeze-dried whole-leaf Aloe arborescens Miller var. natalensis Berger ‍(designated as ‘ALOE’) onN-ethyl-N’-nitro-N-nitrosoguanidine (ENNG)-induced duodenal tumorigenesis in C57BL/ ‍6 mice. Experiment 1: Male mice were given ENNG in drinking water for the first 4 weeks, and then 10% ALOE in ‍basal diet for 16 weeks. Experiment 2: Female mice were given ENNG for 5 weeks, and then 5%, 1% or 0.2% ALOE ‍in the diet were given for 15 weeks. In Experiment 1, the tumor incidence and tumor multiplicity (tumors per mouse) ‍of the duodenum in the ENNG + 10% ALOE group were significantly decreased compared with that in the ENNG ‍alone group. Erythrocyte polyamine levels in the ENNG + 10% ALOE group were also significantly decreased. In ‍Experiment 2, the incidence of duodenal tumors in the ENNG + 5% ALOE group were significantly decreased ‍compared with that in the ENNG alone group. These results indicated that ALOE, especially at 10% in the diet, ‍inhibits ENNG-induced duodenal tumorigenesis in mice.     

Differential proliferative response of gastric mucosa during carcinogenesis induced by N-methyl-N'-nitro-N-nitrosoguanidine in susceptible ACI rats, resistant Buffalo rats, and their hybrid F1 cross

The effect of N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) on the proliferative characteristics of the pyloric epithelium was investigated in ACI and Buffalo rats and their F1 rats, which are susceptible, resistant, and resistant, respectively, to gastric carcinogenesis by this chemical. After injection of bromodeoxyuridine (BrdUrd), DNA synthesizing cells in the pyloric epithelium were stained immunohistochemically with anti-BrdUrd antibody. The average number and range of distribution of cells labeled with BrdUrd in the pyloric glands were significantly larger in ACI rats than in Buffalo or F1 rats after administration of MNNG (83 micrograms/ml in the drinking water) for 2 or 16 weeks. In control rats given tap water for 2 weeks, there was no significant difference in these values in the three groups (Experiment 1). The distribution of cells that were labeled with [methyl-3H]MNNG in the pyloric epithelium was measured by histoautoradiography, and the distribution of cells double labeled with both [methyl-3H]MNNG and BrdUrd was also analyzed. Rats were given 83 micrograms/ml of MNNG in their drinking water for 2 weeks and then received [methyl-3H]MNNG by gavage and an injection of BrdUrd 2 and 1 h, respectively, before sacrifice. The average number of double labeled cells (i.e., replicating cells exposed to MNNG) was significantly larger in ACI rats than in Buffalo or F1 rats. In control rats given tap water without MNNG for 2 weeks, there was no significant difference in these values in the three groups (Experiment 2). Cells double labeled with [methyl-3H]MNNG and BrdUrd are considered to be cells with the potential to establish mutations (cell population at risk of MNNG-induced carcinogenesis). Our results show that, after MNNG treatment, the size of this cell population is larger in susceptible ACI rats than in resistant Buffalo and F1 rats. Thus, differential responses of the gastric mucosa to MNNG may be a key factor in the difference of susceptibility to gastric carcinogenesis between ACI and Buffalo rats.     

Chemopreventive activity of oltipraz against induction of glandular stomach carcinogenesis in rats by N-methyl-N'-nitro-N-nitrosoguanidine [published erratum appears in Carcinogenesis 1998 May;19(5):955]

The modifying effects of oltipraz on induction of glandular stomach carcinogenesis by N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) were investigated in a total of 120 male 6-week-old Wistar rats, divided into six groups. Groups 1-3 (30 animals each) were given 100 p.p.m. MNNG in their drinking water for 10 weeks as an initiation treatment for gastric cancer induction and respectively fed diets supplemented with 0.04%, 0.02% and 0% oltipraz for 12 weeks, starting 1 week before and finishing 1 week after the carcinogen exposure. Groups 4-6 (10 animals each) were similarly treated without the application of MNNG. At the end of the 80th experimental week, all surviving animals were autopsied and examined histopathologically for the existence of gastric proliferative lesions. The incidence and multiplicity of adenocarcinomas were significantly (P < 0.01) lower in group 1 than in group 3. In addition, the multiplicity of atypical hyperplasias in the pyloric region was significantly (P < 0.05) decreased in group 1 as compared with the group 3 value. No gastric proliferative lesions were found in groups 4-6. In an additional short-term experiment, oltipraz significantly reduced cell proliferative activity (P < 0.01) and elevated glutathione levels (P < 0.05) in the glandular stomach mucosa of rats treated with MNNG. Thus our results clearly indicate that oltipraz can inhibit induction of proliferative glandular stomach lesions by MNNG in the rat.      

Sequential histologic changes during gastric carcinogenesis induced by N-methyl-N'-nitro-N-nitrosoguanidine in susceptible ACI and resistant BUF rats

Sequential histologic changes of the stomach during carcinogenesis induced by N-methyl-N'-nitro-N-nitrosoguanidine (MNNG; CAS: 70-25-7) were studied in susceptible ACI and resistant BUF strain rats. Rats were given MNNG at a concentration of 83 micrograms/ml in their drinking water for 32 weeks and then tap water and were sacrificed sequentially between weeks 1 and 57. In ACI rats, erosions, regenerative changes, focal and slightly atypical changes, and diffuse and severe atypical changes were observed sequentially in the pyloric region during the period of MNNG administration, where adenocarcinomas were observed after the cessation of MNNG treatment. In BUF rats, the main histologic changes induced by MNNG were erosions and hyperplasia of the glandular portion of pyloric glands at the margin of erosions. After the cessation of MNNG treatment, the hyperplasia of the pyloric glands subsided and was followed by atrophy of these glands. The results suggested that the responses of the gastric mucosa to MNNG in ACI and BUF rats were qualitatively different.     

Induction of intestinal metaplasia in rats by N-ethyl-N'-nitro-N-nitrosoguanidine but not by sodium hydroxide

Intestinal metaplasia (IM) in the glandular stomach of male Wistar rats induced by oral administration of N-ethyl-N'-nitro-N-nitrosoguanidine (ENNG) and/or intubation of 0.1N sodium hydroxide (NaOH) was studied as follows. Experiment I, sequential study: Rats in group I were given 100 micrograms/ml ENNG in drinking water for 12 weeks. Rats in group II were given 5 ml of 0.1N NaOH by gastric intubation once a week for 12 weeks. Group III control rats were given tap water. Rats were killed from week 1 until week 69 sequentially. IM was first found at week 26 in group I and at week 58 in groups II and III, its incidence being significantly higher in group I than in the other two groups (P less than 0.01), but without any difference between group II and group III. Experiment II, two-stage carcinogenesis: Rats in groups I and II were treated in the same way as in experiment I, while rats in group III were given 100 micrograms/ml ENNG for 12 weeks, followed by 0.1N NaOH once a week for 12 weeks intragastrically. All rats were killed at week 56. The numbers of metaplastic glands in groups I and III were higher than in group II. Gastric carcinomas were induced in all groups of rats treated with ENNG. The results of these two experiments show that IM is effectively induced by a carcinogen but is not enhanced by regeneration induced by alkaline treatment.     

Effects of four antioxidants on N-methyl-N'-nitro-N-nitrosoguanidine initiated gastric tumor development in rats

The effects of antioxidant administration during the post initiation phase of gastric tumor development were investigated in male Wistar rats treated with N-methyl-N'-nitro-N-nitrosoguanidine (MNNG). Animals (20/group) were given MNNG in the drinking water (100 mg/l) for 8 weeks, and for the duration of this treatment were also fed on diet supplemented with 10% sodium chloride. Thereafter, they were divided into 6 groups and were maintained on diet containing either 2% butylated hydroxyanisole (BHA), 1% BHA, 1% butylated hydroxytoluene (BHT), 1% ethoxyquin (EQ) or 1% DL-alpha-tocopherol (alpha-TP) for 32 weeks. A carcinogen control group was fed the basal diet without antioxidant supplementation. The experiment was terminated 40 weeks after the beginning of administration of MNNG and development of gastroduodenal tumors was determined histopathologically. EQ significantly increased the incidence of tumors in the glandular stomach. No modification of tumor development in this region of the organ were observed with 2% BHA, 1% BHA, 1% BHT or 1% alpha-TP, although both 2% BHA and 1% BHA induced and/or promoted tumor development in the forestomach. In addition, nephrocalcinosis was identified only in the kidneys of rats given EQ after MNNG treatment.     

Inhibition of ENNG-induced pyloric stomach and small intestinal carcinogenesis in mice by high temperature- and pressure-treated garlic

High temperature- and pressure-treated garlic (HTPG) has been shown to have enhanced antioxidative activity and polyphenol contents. Previously, we reported that HTPG inhibited 1,2-dimethylhydrazine-induced mucin depleted foci (premalignant lesions) and O6-methylguanine DNA adduct formation in the rat colorectum. In the present study, we investigated the modifying effects of HTPG on N-ethyl-N'-nitro-N-nitrosoguanidine (ENNG)- induced pyloric stomach and small intestinal carcinogenesis in mice. Male C57BL/6 mice were given ENNG (100 mg/l) in drinking water for the first 4 weeks, then a basal diet or diet containing 2% or 5% HTPG for 30 weeks. The incidence and multiplicity of pyloric stomach and small intestinal (duodenal and jejunal) tumors in the 2% HTPG group (but not in the 5% HTPG group) were significantly lower than those in the control group. Cell proliferation of normal-appearing duodenal mucosa was assessed by MIB-5 immunohistochemistry and shown to be significantly lower with 2% HTPG (but again not 5% HTPG) than in controls. These results in dicate that HTPG, at 2% in the diet, inhibited ENNG-induced pyloric stomach and small intestinal (especially duodenal) tumorigenesis in mice, associated with suppression of cell proliferation.