Influences of diethylnitrosamine on longevity of surrounding hepatocytes and progression of transplanted persistent nodules during phenobarbital promotion of hepatocarcinogenesis

The possibility that phenobarbital (PB) selectively promotes liver nodule development by decreasing survival of surrounding hepatocytes previously exposed to diethylnitrosamine (DENA) was evaluated. Livers of F-344 rats were labelled with [3H-methyl]-thymidine (3H-TdR) during developmental or regenerative growth. Neonatal rats given 3H-TdR between days 3 and 12 were subjected at 12 weeks of age to partial hepatectomy (PH) followed 24 hr later by DENA (10 mg/kg) or saline. Subsequent administration of PB (0.1% in drinking water) for 28 weeks reduced total liver label to 46 +/- 10% (saline group) or 40 +/- 4% (DENA group). Adult male rats initiated with DENA (200 mg/kg) and later labelled with 3H-TdR after PH also lost total liver label during 28 weeks' promotion with PB (0.05% in water) at rates similar to those exhibited by noninitiated rats given PB, and by DENA-treated or control rats not given PB. Large persistent (12 weeks) liver nodules generated by DENA in the Solt-Farber model were transplanted as small fragments into the spleens of syngeneic rats previously given 0, 100 or 200 mg/kg of DENA. Subsequent exposure to PB (0.05% in drinking water for 40 weeks) or Aroclor 1254 (6 X 300 mg/kg per month) promoted nodule and cancer development only in livers of DENA-initiated recipients. Surviving transplanted nodules remained as small microscopic clusters even after 40 weeks of promotion. However, PB increased transplant survival (50% vs. 21% in controls) whereas Aroclor reduced it to 8%. These findings indicate that promotion of liver nodules by PB occurs without enhanced mortality of surrounding hepatocytes previously damaged by DENA. They further suggest that promoters such as PB and PCBs do not directly influence the progression of established persistent nodules.     

Phenotypic shift in human differentiated gastric cancers from gastric to intestinal epithelial cell type during disease progression

Background. The phenotypic expression of tumor cells is widely thought to resemble that of the tissue of origin. In the present study, to assess phenotypic changes that occur with disease progression, we investigated human differentiated gastric cancers at different depths of invasion for component cancer cell types. Methods. Using a combined mucin histochemical and immunohistochemical approach, we classified surgical specimens of 301 differentiated gastric cancers into three types: gastric epithelial cell (G) type, intestinal epithelial cell (I) type and mixed gastric and intestinal (GI) type, according to the phenotypic differentiation of the component cancer cells. The relation between the phenotypic type of cancer and their depth of invasion was evaluated. Results. The proportion of G type cancers was 41.4% in early (tumor invasion of mucosa or submucosa) cases, decreasing to 22.2% in advanced (tumor invasion of muscularis propia or deeper) cases, whereas the proportion of I type cancers increased with progressive disease from 23.5% to 31.1% (P < 0.01). Cancers invading the subserosa or deeper included more I type cases and fewer G type than cancers limited to the mucosa (P < 0.01). In most cases of each phenotypic type, intestinal metaplasia was recognized in the surrounding background mucosa, but no clear relation was shown between the phenotype of cancers and the degree of intestinal metaplasia in the background mucosa, suggesting that intestinal metaplasia is not always a preneoplastic lesion. Conclusions. A phenotypic shift from G to I type expression was observed with the progression of human differentiated gastric cancers. Intestinalization may occur independently in cancerous and noncancerous gastric mucosa. Received for publication on May 1, 1998; accepted on Oct. 22, 1998     

Eradication of Helicobacter pylori induces apoptosis and inhibits proliferation of heterotopic proliferative glands in infected Mongolian gerbils

Mongolian gerbils infected with Helicobacter pylori (H. pylori ) develop heterotopic proliferative glands (HPGs) in the glandular stomach submucosa. To investigate the effects of H. pylori eradication on cell turnover in HPGs, three antibiotics, lansoprazole, amoxicillin and clarithromycin, were administered at 50 or 75 weeks after inoculation of H. pylori, and the stomachs were excised for histological examination at 1, 2, 4, 8 or 25 weeks thereafter. The HPGs were classified into gastric type (G-type) and others (GI + I-type), which included both pure intestinal (I-type) and gastric-and-intestinal mixed type (GI-type). Apoptosis and cell proliferation were evaluated by means of TUNEL assay and BrdU labeling, respectively. At 8 weeks post-eradication, apoptotic indices were significantly increased in the eradication group (G-type: 2.5%; GI + I-type: 7.2%) compared to the non-eradication group (G-type: 0.6%; GI + I-type: 2.1%: P < 0.01), while BrdU labeling indices were significantly decreased (G-type: 1.9%; GI + I-type: 6.8% as compared with 4.3% and 13.2%, respectively, P < 0.01 for both). At 25 weeks, the apoptotic indices were similarly higher [G-type: 0.4 (eradication group) vs. 0.2% (non-eradication group); GI + I-type: 5.8 vs. 1.1%, both P < 0.01], and the BrdU labeling indices (G-type: 0.8 vs. 2.2%, P < 0.01; GI + I-type: 5.1 vs. 11%, P < 0.05) continued to be lower in HPGs. Furthermore, there were highly significant reductions in the areas of HPGs at 8 and 25 weeks post-eradication. These findings demonstrated that eradication results in apoptosis and reduction of proliferation of HPGs in H. pylori-infected gerbils, these lesions thus being apparently reversible through regulation of cell kinetics.     

Dose responses of five hepatocarcinogens for the initiation of rat hepatocarcinogenesis

Using the Solt and Farber model (Nature, 263 (1976) 701), dose-response relationships between initiating agents and the induction of hyperplastic nodules in rat liver were investigated. Male Fischer 344 rats were initiated by a single application of 1 of 5 carcinogens at 3 doses: 200.0, 50.0 and 12.5 mg/kg of diethylnitrosamine (DEN); 1.00, 0.25 and 0.062 mg/kg of aflatoxin B1 (Af-B1); 60.0, 15.0 and 3.75 mg/kg of N-2-fluorenylacetamide (2-FAA); 600.0, 150.0 and 37.5 mg/kg of 3'-methyl-4-dimethylaminobenzene (3'-me-DAB); 40.0, 10.0 and 2.5 mg/kg of dimethylnitrosamine (DMN) and their vehicles. Two weeks after initiation, animals were placed on a 0.02% 2-FAA diet for 2 weeks. Partial hepatectomy was performed at the end of the third week of the experiment. All rats were killed 4 weeks after the initiation, and the hyperplastic nodules of the liver were counted and their areas measured. Dose responses were observed in both numbers and areas of hyperplastic nodules per unit area of sections with all of the 5 carcinogens examined.     

Helicobacter pylori infection enhances glandular stomach carcinogenesis in Mongolian gerbils treated with chemical carcinogens

Helicobacter pylori (Hp) is thought to be a stomach carcinogen from epidemiological findings. To determine the effects of infection with the bacteria on experimental carcinogenesis, a study of the glandular stomach of Mongolian gerbils (MGs) was performed. Male MGs were treated with N-methyl-N'-nitro-N-nitrosoguanidine followed by inoculation with Hp or infected with Hp followed by N-methyl-N'-nitro-N-nitrosoguanidine administration. Animals were killed at week 50, and their excised stomachs underwent microbiological and histopathological examinations. In addition, a serological investigation was performed. The incidences of adenocarcinomas were significantly higher in animals treated with 60 or 300 p.p.m. N-methyl-N'-nitro-N-nitrosoguanidine for 10 weeks followed by Hp inoculation or Hp followed by 20 p.p.m. N-methyl-N'-nitro-N-nitrosoguanidine for 30 weeks than in the respective controls. Moreover, tumour-bearing animals had higher titres of anti-Hp antibodies than tumour-free animals. Of interest was the finding that a dose of 100 p.p.m. N-methyl-N'-nitro-N-nitrosoguanidine given to infected gerbils eradicated the Hp in about half the animals, with a concomitant reduction in the promoting effect. No tumours were found in animals infected with Hp without N-methyl-N'-nitro-N-nitrosoguanidine or non-treated gerbils. Hp infection enhances glandular stomach carcinogenesis in MGs treated with N-methyl-N'-nitro-N-nitrosoguanidine. Animals with high titres of anti-Hp antibodies are at greatest risk of developing neoplasms.     

Rare mutations of p53, Ki-ras, and beta-catenin genes and absence of K-sam and c-erbB-2 amplification in N-methyl-N'-nitro-N-nitrosoguanidine-induced rat stomach cancers.

Rat stomach cancers induced by N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) have been widely used as a model for human stomach cancers of the differentiated type. However, there has been little information regarding their molecular basis. In this study, we examined the genetic alterations reported in human stomach cancers in 10 rat stomach cancers that had been induced in male ACI/N rats by administering MNNG in the drinking water. One of the 10 cancers had a mutation of the p53 gene at the second position of codon 171 (Val --> Glu). However, none of the 10 cancers had mutations in codons 12, 13, or 61 of Ki-ras or in the N-terminal phosphorylation sites of the beta-catenin gene. Southern blot analysis showed no amplification of K-sam or c-erbB-2 in the seven cancers examined. Finally, we searched for microsatellite alterations in 12 loci in nine cancers, but no alterations were observed. As these genetic alterations are observed in only a minor fraction of human stomach cancers, further analysis of genetic and epigenetic alterations in MNNG-induced rat stomach cancers is needed to disclose the major mechanisms of stomach carcinogenesis.     

Gastric and intestinal phenotypes and histogenesis of advanced glandular stomach cancers in carcinogen-treated, Helicobacter pylori-infected Mongolian gerbils

The Helicobacter pylori-infected Mongolian gerbil (MG) has been established as an appropriate animal model for studies of stomach cancer development. However, there have hitherto been no data on the phenotypic classification of glandular stomach cancers in H. pylori-infected and non-infected MG. We therefore examined the phenotypes of 50 and six advanced glandular stomach cancers in H. pylori-infected and non-infected MG, respectively, as well as adjacent non-neoplastic mucosa, using several gastrointestinal epithelial phenotypic markers. The lesions were divided phenotypically into 21 gastric, 24 gastric-and-intestinal mixed, four intestinal and one null types, with 90.0% of the lesions harboring gastric elements and 56.0% demonstrating intestinal phenotypic expression in H. pylori-infected MG. All six lesions were classified as gastric type in non-infected MG. There was no clear correlation with the presence of intestinal metaplasia in surrounding mucosa. In conclusion, our data suggest that most advanced adenocarcinomas retain a gastric cellular phenotype in the glandular MG stomach. Thus, it might be proposed that intestinal metaplasia is a paracancerous phenomenon rather than a premalignant condition. H. pylori infection may trigger intestinalization of both stomach cancers and non-neoplastic mucosa.     

Helicobacter pylori infection stimulates intestinalization of endocrine cells in glandular stomach of Mongolian gerbils

Intestinal metaplasia has been investigated extensively as a possible premalignant condition for stomach cancer but its pathogenesis is still not fully understood. In the present study, we examined the relationship between endocrine and mucous cell marker expression periodically after Helicobacter pylori infection in the Mongolian gerbil model. The numbers of chromogranin A (CgA)-positive, gastrin-positive and gastric inhibitory polypeptide (GIP)-positive cells in H. pylori-infected groups was increased significantly compared with the non-infected case. However, CgA-positive and gastrin-positive cells then decreased from 50 through 100 experimental weeks after H. pylori infection, whereas GIP-positive cells increased. Coexistence of gastrin-positive and GIP-positive cells was detected in the same gastric and intestinal mixed phenotypic glandular-type glands. In conclusion, the endocrine cell phenotype is in line with that of the mucous counterpart in the glands of H. pylori-infected Mongolian gerbil stomach, supporting the concept that development of intestinal metaplasia is due to the abnormal differentiation of a stem cell.