p53 knockout mice (-/-) are more susceptible than (+/-) or (+/+) mice to N-methyl-N-nitrosourea stomach carcinogenesis

Mutations of the p53 tumor suppressor gene constitute one of the most frequent molecular changes in a wide variety of human cancers. Mice deficient in p53 have recently attracted attention for their potential to identify chemical genotoxins. In this study we have investigated the susceptibility of p53 nullizygote (-/-), heterozygote (+/-) and wild-type (+/+) mice to N:-methyl-N:-nitrosourea (MNU) gastric carcinogenesis. p53 knockout mice were treated with 30 p.p.m. MNU in the drinking water 1 week on and 1 week off and killed after 5 weeks. The numbers of pepsinogen-altered pyloric glands (PAPG), putative preneoplastic lesions, were 1.8, 1.7 and 22.6 in p53 (+/+), (+/-) and (-/-) mice, respectively. In a 15 week experiment, adenomas were found in 0 of 19 (+/+) (0%), 2 of 21 (+/-) (9.5%) and 6 of 10 (-/-) (60.0%) animals. Also, one well-differentiated adenocarcinoma was observed in a p53 (-/-) mouse. After 40 weeks treatment with 120 or 30 p.p.m. MNU there was no significant difference in the incidence of gastric tumors between p53 (+/+) and (+/-) mice. However, mortality from carcinogen-induced lymphomas, leukemias and sarcomas was very much greater in the latter group. Homozygous knockout animals could not be maintained long term. PCR-single strand conformation polymorphism analysis of exons 5-8 of the p53 gene of DNA extracts from 68 gastric tumors consisting of 16 and 20 30 p.p.m. MNU-treated p53 (+/+) and (+/-) mice and 14 and 18 120 p.p.m. MNU-treated p53 (+/+) and (+/-) mice demonstrated no mutations. These results suggest that p53 may not be a direct target of MNU but rather play an important role as a gatekeeper in mouse stomach carcinogenesis induced by this direct acting agent.     

The effects of methylnitrosourea (MNU) on natural killer (NK) cell cytotoxicity and cytokine production in rats

Eight-week-old male Sprague–Dawley rats were exposed tothe carcinogen methylnitrosourea (MNU) via gastric intubationat doses of either 10 or 20 mg/kg body wt. Rats were treatedonce a week for 4 weeks, then once every 2 weeks for 1 month,for a total of 6 treatments. MNU was found to exert no consistentsignificant immunosuppressive effects in vivo as measured byspleen natural killer (NK) cell cytotoxicity, interleukin-2(IL-2) production by splenic lymphocytes and prostaglandin E₂(PGE₂) production by adherent peritoiieal macrophages. In contrast,splenic NK cell cytotoxicity and IL-2 production of MNU-treatedrats were actually elevated at several of the later samplingperiods. PGE₂ production was also elevated in MNU-treated ratsin the later sampling periods. Body weights of MNU-treated ratswere markedly decreased as early as 4 weeks following the initialMNU treatment. This suppression persisted throughout the study.The most dramatic change in organ weights was seen in the thymus.Thymus weights of all MNU-treated rats were significantly decreased1 day after treatment and persisted for 4 weeks. By the 60 daysampling period, thymus weights were not significantly differentfrom controls. However, by 120 and 180 days, thymus weightsagain were significantly lowered In those rats receiving MNU.These changes in thymus weights were accompanied histologicallyby initial cortical thinning and progressive loss of corticalthymocytes followed by the appearance of hyperplastic and neoplasticcells. It thus appears that the carcinogenic effect of MNU isnot related to a depression of the Immune surveillance system,at least as measured by NK cell activity.     

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.     

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.     

Effect of dietary alfalfa, pectin, and wheat bran on azoxymethane-or methylnitrosourea-induced colon carcinogenesis in F344 rats.

The effect of dietary alfalfa, pectin, and wheat bran on colon carcinogenesis was studied in female inbred F344 rats. Weanling rats were fed semipurified diets containing 0 or 15% alfalfa, pectin, or wheat bran. At 7 weeks of age, all animals except controls were given azoxymethane (AOM) sc at a dose rate of 8 mg/kg body weight/week for 10 weeks or methylnitrosourea (MNU) intrarectally at a dose rate of 2 mg/rat twice a week for 3 weeks. The AOM-treated group was autopsied 40 weeks and the MNU-treated group 30 weeks after the first injection of the carcinogen. No tumors were observed in the colon or other organs of untreated rats fed the various diets. The animals fed the alfalfa diet and treated with MNU had a higher incidence of colon tumors than did those fed the control diet or the diets containing pectin or wheat bran. The incidence of MNU-induced colon tumors did not differ between the animals fed the control diet or the diets containing pectin or wheat bran. However, the incidence of AOM-induced colon tumors in rats fed diets containing pectin or wheat bran was lower than that in rats fed the control diet or the alfalfa diet. These results thus indicate that the effect of fiber in colon carcinogenesis depends on the type of fiber and, possibly, the fiber's mode of action.     

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.     

Natural killer activity in a medium-term multi-organ bioassay for carcinogenesis.

Natural killer (NK) cell activity was evaluated after the initiation and promotion steps in a medium-term multi-organ bioassay for carcinogenesis. NK cell activity was assessed in vitro by Cr51 release assay at the 4th and 30th weeks of the experiment. Male Wistar rats were sequentially initiated with N-diethylnitrosamine (DEN i.p.), N-butyl-N-(4-hydroxybutyl)nitrosamine (BBN drinking water), N-methyl-N-nitrosourea (MNU i.p.), dihydroxy-di-N-propylnitrosamine (DHPN drinking water) and N,N'-dimethylhydrazine (DMH s.c.) at subcarcinogenic doses for 4 weeks (DMBDD initiation). One group was evaluated at the 4th week and the other was maintained without any further treatment until the 30th week. Two initiated groups were exposed through the diet to 2-acetylaminofluorene (2-AAF) or phenobarbital (PB), from the 6th until the 30th week. Five additional groups were studied to evaluate the effects of each initiator on NK activity. All groups submitted to initiation only, initiation plus promotion, or promotion only, developed significantly more preneoplastic lesions than the untreated control group. The main target organs for tumor development in the initiated animals were the liver and the colon, irrespective of treatment with 2-AAF or PB. NK cell activity was not affected by exposure to genotoxic carcinogens after initiation, at the 4th week. Treatments only with PB or 2-AAF did not change NK cell activity. However, decreased NK cell activity was registered in the group only initiated with DMBDD and in the group given DMBDD+2-AAF. This late depression of NK cell activity at the 30th week could be related to the production of suppressing molecules by the tumor cells.     

Natural Killer Activity in a Medium-term Multi-organ Bioassay for Carcinogenesis

Natural killer (NK) cell activity was evaluated after the initiation and promotion steps in a medium-term multi-organ bioassay for carcinogenesis. NK cell activity was assessed in vitro by Cr⁵¹ release assay at the 4th and 30th weeks of the experiment. Male Wistar rats were sequentially initiated with N-diethylnitrosamine (DEN i.p.), N-butyl-N-(4-hydroxybutyl)nitrosamine (BBN drinking water), N-methyl-N-nitrosourea (MNU i.p.), dihydroxy-di-N-propylnitrosamine (DHPN drinking water) and N, N'-dimethylhydrazine (DMH s.c.) at subcarcinogenic doses for 4 weeks (DMBDD initiation). One group was evaluated at the 4th week and the other was maintained without any further treatment until the 30th week. Two initiated groups were exposed through the diet to 2-acetylaminofluorene (2-AAF) or phenobarbital (PB), from the 6th until the 30th week. Five additional groups were studied to evaluate the effects of each initiator on NK activity. All groups submitted to initiation only, initiation plus promotion, or promotion only, developed significantly more preneoplastic lesions than the untreated control group. The main target organs for tumor development in the initiated animals were the liver and the colon, irrespective of treatment with 2-AAF or PB. NK cell activity was not affected by exposure to genotoxic carcinogens after initiation, at the 4th week. Treatments only with PB or 2-AAF did not change NK cell activity. However, decreased NK cell activity was registered in the group only initiated with DMBDD and in the group given DMBDD+2-AAF. This late depression of NK cell activity at the 30th week could be related to the production of suppressing molecules by the tumor cells.     

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.     

Prevention and treatment of primary intestinal tumors in rats by piroxicam

Over 90% of methylazoxymethanol acetate-treated male Lobund Sprague-Dawley rats developed intestinal tumors within 20 weeks; the incidence and numbers of tumors remained basically the same at 40 weeks. However, at week 40 the numbers of large tumors (greater than 0.5 cm in diameter) were increased. At week 40, tumors in methylazoxymethanol acetate-inoculated rats that had been treated with piroxicam (approximately 2.3 mg/day) from week 1 or from week 20 (after tumors had developed) were significantly reduced in numbers, but many large tumors persisted. Rats treated with piroxicam up to 40 weeks carried 0.6 tumor/rat compared with 2.7 tumors/rat among untreated controls (P less than 0.0001). Rats at week 20 had developed 2.8 tumors/rat and rats treated with piroxicam from week 20 to week 40 carried 1.4 tumors/rat (P less than 0.007). Most of the tumors in the piroxicam-treated rats showed evidence of histological differentiation.     

A COX-2 inhibitor prevents the esophageal inflammation-metaplasia-adenocarcinoma sequence in rats

Barrett's esophagus (BE) and esophageal adenocarcinoma (ADC) is associated with reflux of duodenal contents. Cyclooxygenase (COX)-2 is over-expressed in BE and ADC, and supposedly contributes to esophageal carcinogenesis. The aim of this study is to investigate what effect a COX-2 inhibitor has on esophageal adenocarcinogenesis in rats. A series of 90 rats underwent a duodenoesophageal reflux procedure and were divided into 2 groups. One group was given commercial chow (control group), and the other was given experimental chow containing celecoxib (celecoxib group). The animals were sacrificed sequentially, at the 10th, 20th, 30th and, finally, 40th week after surgery, and their esophagi were examined. In the control group, esophagitis, columnar-lined epithelium (CLE) and ADC were first observed at the 10th week, 20th week and 30th week, respectively. Their incidences sequentially increased and at the 40th week reached 100, 89 and 47%, respectively. In the celecoxib group, the esophagitis was mild and the incidence of CLE was significantly lower at each week (P < 0.001), compared with the control group, and ADC was not observed throughout the experiment (P < 0.05). COX-2 expression was observed predominantly in the stroma of inflamed esophageal epithelia, and up-regulated at the 10th and 20th week (P < 0.05, respectively). PGE2 level and proliferative activity were also up-regulated in both groups, but they were lower in the celecoxib group than in the control group (P < 0.05). Apoptosis was observed to increase with celecoxib treatment (P < 0.05). Celecoxib is effective in preventing CLE and ADC by suppressing esophagitis in rats.     

Antioxidative and Modifying Effects of a Tropical Plant Azadirachta indica (Neem) on Azoxymethane-induced Preneoplastic Lesions in the Rat Colon

The purpose of the present study was to examine whether Neem leaf (Azadirachta indica) has short-termchemopreventive effects on endpoint preneoplastic lesions involved in rat colon carcinogenesis and might also exertantioxidative activity. Forty- two male F344 rats were randomly divided into 6 experimental groups. Groups 1 to 4were given a subcutaneous injection of azoxymethane (AOM, 20 mg/kg body weight) once a week for 2 weeks.Starting one week before the first injection of AOM, rats in groups 2 to 4 received an aqueous extract of Neem leaf(20, 100, and 250 mg/kg, respectively) by gavage 3 times per week, for 5 weeks. Rats in group 5 also were given theNeem extract by gavage feeding 3 times per week for 5 weeks, while group 6 served as untreated controls. Theexperiment was terminated 5 weeks after the start. Dietary feeding of the Neem extract at all dose levels significantlyinhibited the induction of aberrant crypt foci (ACF) (P<0.0002), when compared to the AOM-treated group (group1). In groups 2 to 4, treatment of rats with the Neem extract also significantly decreased the proliferating cell nuclearantigen (PCNA) labeling indices (P<0.0006) of colon epithelium and ACF. Moreover, the Neem extract also showedantioxidative activity. The finding that dietary Neem has possible chemopreventive effects in the present short-termcolon carcinogenesis bioassay suggests that longer-term exposure may cause suppression of tumor development.     

Attenuation by d-limonene of sodium chloride-enhanced gastric carcinogenesis induced by N-methyl-N'-nitro-N-nitrosoguanidine in Wistar rats.

The effects of prolonged administration of d-limonene, a monocyclic monoterpene, on sodium chloride-enhanced induction of gastric carcinogenesis by N-methyl-N'-nitro-N-nitrosoguanidine, the labeling and apoptotic indices, and ornithine decarboxylase (ODC) activity of gastric cancers were investigated in Wistar rats. After 25 weeks of carcinogen treatment, rats were given chow pellets containing 10% sodium chloride and 1% limonene ad libitum. In week 52, the incidence of gastric cancers, the labeling index and ODC activity were significantly higher and the apoptotic index was significantly lower in rats given sodium chlolide than in untreated control rats. However, in rats given both sodium chloride and d-limonene, the incidence of gastric cancers, the labeling index and ODC activity were significantly lower and the apoptotic index was significantly higher than in rats given sodium chloride alone. Our findings suggest that limonene attenuates the gastric carcinogenesis enhanced by sodium chloride via increased apoptosis and decreased ODC activity in gastric cancers.     

Chemoprevention of glandular stomach carcinogenesis through duodenogastric reflux in rats by a COX-2 inhibitor

Duodenogastric reflux (DGR) causes glandular stomach carcinogenesis in rats without carcinogens. We aimed to investigate how this carcinogenesis might be prevented by a selective COX-2 inhibitor, meloxicam. A series of 188 Fisher 344 rats underwent a surgical DGR procedure and were divided into 2 groups. One group was given commercial chow (control group), and the other an experimental chow containing meloxicam [0.3 mg/kg bw/day] (meloxicam group). The animals were sequentially sacrificed at weeks 20, 30, 40, 50 and 60 after surgery. The stomachs were removed and examined for the presence of carcinoma, incidence of reflux-induced morphologic changes, COX-2 expression and its activity. Adenocarcinoma in the glandular stomach developed in 7 of 21 animals (33%) in the control group at week 60, but none of 20 (0%) in the meloxicam group (p < 0.01). Moreover, reflux-induced gastritis was definitely alleviated in the meloxicam group compared with the control group. COX-2 immunoreactivity was predominantly detected in stromal cells such as macrophages and fibroblasts. Compared with nonsurgical rats, RNA expression of COX-2 in the mucosa increased, reaching peak at an early phase of week 20 in both groups (p < 0.005). Expression of microsomal prostaglandin E synthase-1 was lower in the meloxicam group than in the control group. PGE(2) production was significantly suppressed throughout the experiment in the meloxicam group compared with the control group (p < 0.01). Gastric carcinogenesis via duodenal reflux was mediated by the COX-2 pathway in rats. Administration of meloxicam prevented this carcinogenesis by suppressing the inflammatory process.     

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.     

Enhanced induction of gastric carcinogenesis by N-methyl-N'-nitro-N-nitrosoguanidine in deoxycorticosterone acetate-NaCl hypertensive rats and its inhibition by potassium chloride

The effect of s.c. administration of deoxycorticosterone acetate (DOCA) plus p.o. treatment with NaCl solution on gastric carcinogenesis induced by N-methyl-N'-nitro-N-nitrosoguanidine and the effect of p.o. potassium supplementation on the enhanced induction of gastric carcinogenesis in DOCA-NaCl rats were investigated in Wistar rats. After 25 weeks of p.o. treatment with the carcinogen, rats received s.c. injections of DOCA (50 mg/kg) twice a week and were given 1% NaCl solution with and without 1% KCl as drinking water. In Week 52, the blood pressure, the incidence of gastric cancer, and the number of cancers per rat were significantly greater in DOCA-NaCl rats than in the untreated group. Prolonged p.o. treatment of DOCA-NaCl hypertensive rats with potassium significantly reduced their blood pressure, the incidence of gastric cancers, and their number per rat. All gastric tumors were in the glandular portions of the stomach. The norepinephrine concentration in the gastric wall and the labeling indices of gastric mucosa were significantly greater in DOCA-NaCl hypertensive rats than in the untreated group, but p.o. potassium supplementation significantly reduced the norepinephrine concentration in the gastric wall and the labeling indices of the gastric mucosa in DOCA-NaCl rats. Thus, administration of DOCA and NaCl increased the norepinephrine concentration in the gastric wall and promoted gastric carcinogenesis, and p.o. potassium supplementation decreased the norepinephrine concentration in the gastric rats. Inasmuch as the norepinephrine concentration has been used as a marker of sympathetic nervous activity, these findings suggest that the sympathetic nervous system plays an important role in gastric carcinogenesis, probably associated with cell proliferation of antral epithelial cells.     

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.     

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.     

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.     

Inhibitory effects of ursodeoxycholic acid on N-methylnitrosourea-induced colon carcinogenesis and colonic mucosal telomerase activity in F344 rats.

Bile acids are known to promote colon carcinogenesis. However, one study showed that ursodeoxychlic acid (UDCA) prevented azoxymethane-induced rat colon tumorigenesis. The aim of the present study with 3 sets of experiments was to explore the inhibitory effect of UDCA supplemented in the diet on colon carcinogenesis induced by the intrarectal administration of N-methylnitrosourea (MNU) in F344 rats. In experiment I, 5 rats per group were fed a diet supplemented with 0% (control), 0.4%, 0.08% or 0.016% UDCA or chenodeoxycholic acid (CDCA) for 5 weeks after receiving 3 intrarectal doses of 4 mg MNU in week 1. The formation of colonic aberrant crypt foci (ACFs, preneoplastic lesions) at week 6 showed a 24% and 23% reduction in the 0.4% and 0.08% UDCA groups, respectively, as compared to the control group, while it increased for the 0.4% and 0.08% CDCA groups, and was unaffected in the 0.016% UDCA and CDCA groups. In experiment II based on the results of experiment I, all rats received an intrarectal dose of 2 mg MNU 3 times a week for 3 weeks, and then were administered with 0%, 0.4% or 0.08% UDCA for 27 weeks. At week 30, the incidence of colon tumors in the UDCA groups was significantly lower than that in the control group: 20/50 (40%) and 9/25 (36%) vs. 17/25 (68%). The number of large-sized ACFs with 4 or more ACs showed a 47% and 59% reduction in the normal-appearing mucosa in the UDCA groups as compared to the control group, while the number of small-sized ACFs with 1-3 ACs was similar in all groups. The normal-appearing mucosa showed a noticeable level of telomerase activity (semiquantitative PCR-based TRAP assay) in the control group, and significantly reduced levels in the UDCA groups compared to the control group: 19.8 and 32.7 vs. 71.0 TPG unit in mean value. The colon tumors showed a high level of enzyme activity in both the control and UDCA groups. In experiment III, 6 rats per group were fed a diet supplemented with 0%, or 0.4% UDCA or CDCA for 5 weeks after receiving 3 intrarectal doses of 4 mg MNU in week 1. Two control groups did not receive any treatment with MNU and bile acids. The MNU-treated groups showed significantly elevated levels of colonic mucosal telomerase activity at week 6 as compared to the control group (6.5 TPG unit in mean value). It was noted that both UDCA and CDCA administration reduced the enzyme activity as compared to the group with MNU treatment alone: 24.7 and 25.2 vs. 40.1 TPG unit in mean value. Thus, the present study suggested that orally administered UDCA inhibited the growth of ACFs and the development of carcinomas in the colon of rats treated with MNU. Also, UDCA may suppress MNU-induced telomerase activation in normal-appearing but ACF-containing colon mucosa, and its mechanism appears to be different from that responsible for the anti-tumor promoting action of UDCA.