Key words: Gastric carcinogenesis - NaC1 - N-Methyl-N'-nitro-N-nitrosoguanidine - Rat stomach

Transcription

1 [Gann, 75, ; June, 1984] EFFECTS OF SODIUM CHLORIDE, SACCHARIN, PHENOBARBITAL AND ASPIRIN ON GASTRIC CARCINOGENESIS IN RATS AFTER INITIATION WITH N-METHYL-N'-NI TRO-N-NI TROSOGUAN I DINE Michihito TAKAHASHI, Takeshi KOKUBO, Fumio FURUKAWA, Yuji KUROKAWA and Yuzo HAYASHI Department of Pathology, National Institute of Hygienic Sciences, Kamiyoga , Setagaya-ku, Tokyo 158 Sodium chloride, saccharin sodium, phenobarbital sodium and aspirin were tested for tumor-promoting activity in the glandular stomach of rats after initiation with N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) coupled with administration of a high salt diet. Male outbred Wistar rats were given MNNG in the drinking water (100 mg/liter) for 8 weeks, and during this period they were fed on diet supplemented with 10% sodium chloride. Thereafter, they were divided into 5 groups and fed on the basal diet or one of various diets supplemented with 10% sodium chloride, 5% saccharin, 0.05% phenobarbital or 1% aspirin until the end of the experiment. All animals were killed at the 40th experimental week for necropsy and histological examination. The incidence of adenocarcinoma was increased in the group given sodium chloride following initiation by MNNG and sodium chloride as compared with the group given MNNG and sodium chloride initiation only, but not significantly. However, the incidence of preneoplastic hyperplasia was significantly increased in this group. Saccharin also enhanced the development of adenocarcinomas of the glandular stomach. The results indicated that dietary administration of sodium chloride or saccharin after MNNG tends to promote tumor development. Phenobarbital or aspirin did not enhance tumor development, aspirin in fact rather showing a tendency to decrease the tumor incidence. Key words: Gastric carcinogenesis - NaC1 - N-Methyl-N'-nitro-N-nitrosoguanidine - Rat stomach Epidemiologic data show that gastric cancer incidence is high in Japan" and eastern Europe, while its incidence has dramatically decreased in the United States." An epidemiological survey carried out on immigrants from Japan to the United States suggested that changes in their life style, especially in the diet, were responsible for the decrease in the incidence of stomach cancer observed in the second generation." These data suggest that dietary habit is a major environmental factor in gastric carcinogenesis.e' Furthermore, Doll and Peto" and Wynder and Gori28' have indicated, after epidemiological surveys of cancer in the United States, that, among the tumor sites associated with certain diets, gastrointestinal carcinomas have an especial claim to being an example where food-borne factors play an important role. Epidemiologic studies have repeatedly let to the conclusion that an increased sodium chloride content in the diet results in an increased incidence of gastric tumors.10' Sato et al.11, 20' and Hirayama8" 9' have found a close association of excess intake of salted foods with stomach 494 Gun

2 TUMOR PROMOTION IN GASTRIC CARCINOGENESIS cancer. Sodium chloride is thought to play a promoting role in the etiology of gastric cancer. Although attempts to induce gastric cancer in animals by feeding salted foods alone were unsuccessful,"' evidence was obtained suggesting a possible role for sodium chloride in experimental gastric carcinogenesis. We demonstrated experimentally in rats that an excess intake of sodium chloride increased tumor incidences in the forestomach induced by 4-nitroquinoline 1-oxide (4-NQO) and in the glandular stomach induced by N - methyl - N' - nitro - N - nitrosoguanidine (MNNG) when given to rats concomitantly with the carcinogen.25, 271 However, those results failed to establish a role for sodium chloride in the promotion of gastric carcinogenesis. Other previous work 21, 251 also failed to demonstrate promoter action of sodium chloride in the two-stage process of gastric carcinogenesis initiated by MNNG, probably because of inappropriate experimental designs. Therefore, utilizing a two-stage carcinogenesis model with MNNG plus sodium chloride treatment as the initiator, we examined the promoting effects of sodium chloride in gastric carcinogenesis and compared the results with the actions of saccharin sodium, a known bladder promoter '21 phenobarbital, a hepatic promoter,"" and aspirin, a mucosal damaging agent.") MATERIALS AND METHODS Seven-week-old male Wistar rats (Shizuoka Laboratory Center, Shizuoka) were housed in plastic cages, 5 rats/cage and maintained under constant conditions (23±2 temperature, 55±5% relative humidity and a 12-hr-light-12-hr-dark cycle). Two hundred and twenty rats were divided into 10 groups (40 rats in group 1 as a control given the initiation procedure alone and 20 rats in each of the other groups). The animals in groups 1 to 5 were given MNNG (Aldrich Chemicals, Inc., U.S.A.) continuously in their drinking water at a concentration of 100 mg/liter and were simultaneously administered the sodium chloride diet, a stock diet (Oriental MF, Oriental Yeast Co., Ltd., Tokyo) supplemented with 10% sodium chloride (Wako Pure Chemical Ind. Ltd., Tokyo), for the first 8 weeks. After this 8-week treatment period, the carcinogen solution was replaced by tap water. Groups 6 to 10 received neither MNNG nor sodium chloride diet in the first 8 weeks of the experiment and served as negative controls maintained on stock diet alone. The animals in groups 1 and 6 were control groups which received the stock diet without any supplementation during the promotion stage. Animals in groups 2 and 7 were fed the 10% sodium chloride diet during the promotion stage (week 8 to week 40). Animals in groups 3 and 8 were maintained on diet supplemented with 5% saccharin (Oriental Pharmaceutical & Synthetic Chemical Co., Ltd., Osaka), while groups 4 and 9 received 0.05% phenobarbital (Iwaki Pharmaceutical Co., Ltd., Tokyo) diet, and groups 5 and 10 received the 1% aspirin (Tokyo Kasei Kogyo Co., Ltd., Tokyo) diet during the promotion stage. All animals had free access to food and water. The experimental animals were examined and weighed weekly. Necropsies were performed on all animals which died or which were killed after becoming moribund, except for a few animals in each group which died in the early stages of the experiment. At the end of the 40th experimental week, all surviving animals were sacrificed and autopsied. Animals which survived for more than 30 weeks were included in the effective numbers of rats. The stomach and other organs were subjected to careful macroscopic examination and then fixed in 10% buffered formalin, prepared for histology by routine methods, and stained with hematoxylin and eosin (H-E). The tumor incidences were analyzed by Fisher's exact probability test and/or the chi-squared test. RESULTS During the initiation period the growth of animals was significantly affected by the presence of MNNG in the drinking water and simultaneous administration of the sodium chloride-supplemented diet. Animals receiving MNNG (groups 1 to 5) showed a marked repression of weight gain (Fig. 1) when compared to MNNG-untreated animals (groups 6 to 10). After cessation of MNNG administration (at 8 weeks), these weight-deficient animals showed a com- 75(6)

3 M. TAKAHASHI, ET AL. Fig. 1. Mean body weight gain of rats fed various diets beginning after the 8th experimental week Animals in groups I to 5 were treated with both MNNG and sodium chloride whereas groups 6 to 10 were maintained on control diet as negative controls. Fig. 2. Preneoplastic hyperplasia showing irregularly arranged glands. H-E. X Gam

4 TUMOR PROMOTION IN GASTRIC CARCINOGENESIS pensatory increase in growth rate although their weights at the termination of the experiment were still lower than those in MNNG-untreated groups. Body weight gains of rats in the different MNNG-treated groups were similar to each other and to the control (group 1) during the promotion period with the exception that in rats receiving the aspirin - supplemented diet (group 5) a marked retardation of growth as compared to the other groups (groups I to 4) was observed. A similar response was observed in group 10 when compared with groups 6 to 9. Gastric tumors were predominantly located in the pyloric region and were grossly observed as plaque-like or nodular lesions with ulceration. MNNG-induced histological lesions in the glandular stomach have been classified simply into 2 categories (Table I) : preneoplastic hyperplasias and adenocarcinomas. Preneoplastic hyperplasias were defined as proliferative, noninvasive mucosal lesions which were located predominantly in the pyloric region. These lesions have previously been known as adenomatous hyperplasia or adenoma 211 but, since they are considered to be the earliest manifestation of carcinogen-induced growth abnormalities in the stomach, the term preneoplastic seems appropriate (Fig. 2). Adenocarcinomas showed irregular glandular structures lined by moderately atypical, darkly stained, or mucin-producing columnar epithelium with enlarged nuclei. Most were relatively well-differentiated and composed of a typical glandular structure with a tubular pattern and cellular or structural atypism. The degree of invasiveness of adenocarcinomas varied from submucosal penetration to involvement of the deeper layers of the stomach wall. No metastasis was found. Pyloric metaplasias are focal abnormalities of the fundic mucosa and represent a type of regenerative lesion after carcinogeninduced damage to the fundic mucosa. Pyloric metaplasias were present in the stomach of many MNNG-treated rats and were apparently unrelated to cancers or sodium chloride; therefore, their incidence is not reported in the table. No lesions were apparent in the fundic region. Table I summarizes the occurrence of gastroduodenal tumors in the various experimental groups (data for groups 6 to 10 are omitted). The incidence of gastric adenocarcinoma tended to increase in sodium chloride-treated rats (group 2) and saccharin-treated rats (group 3) compared to basal diet rats (group 1). In addition, these groups showed higher incidences of preneo- Table I. Promotion Effects of Various Chemicals on Gastroduodenal Carcinogenesis Initiated by MNNG and NaCI Treatment in Male Wistar Rats *P<0.05. a) I fibrosarcoma and 1 hemangiosarcoma. b) Hepatocellular carcinoma. Data for groups 6-10 are ommitted. 75(6)

5 M. TAKAHASHI, ET AL. plastic hyperplasias, the difference being significant (P<0.05) in the case of the sodium chloride-treated group. However, the numbers of adenocarcinomas and preneoplastic hyperplasias were not increased in the rats fed phenobarbital or aspirin as compared with the controls. The incidence of duodenal adenocarcinoma did not differ among the treated and control groups although there seemed to be a slight increase in the sodium chloride-treated group. As compared to normal gastric mucosa, diffuse deep gastric pits with a clearly increased number of mucous neck cells in the fundic mucosa were the major finding in the sodium chloride-treated rats (groups 2 and 7). Diffuse alterations in the fundic mucosa were also observed in saccharin-treated rats (groups 3 and 8) where the thickness of the zone of proliferating cells was clearly increased. Histological examination of the mucosa of aspirin-treated animals (groups 5 and 10) revealed the presence of focal erosions or even ulcers, penetrating to a depth of up to the base of the glandular mucosa, flanked by apparently normal tissue. In the phenobarbital-treated rats (groups 4 and 9), the glandular stomach mucosa were intact. Adenocarcinomas of the duodenum were found mostly within 10 cm of the pyloroduodenal junction as localized swellings. The lumenal surface of the tumors was papillary, nodular, or ulcerative. All duodenal adenocarcinomas were well differentiated and of tubular structure. Although malignant tumors outside of the gastroduodenal tract were rare, one hepatocellular carcinoma was found in the initiation plus phenobarbital-treated group (group 4). DISCUSSION Tumor promotion has been widely investigated in several organs such as the skin, liver, kidney, urinary bladder, mammary gland, and thyroid. In the stomach, although croton oil has been experimentally shown to be a promoter in rats by Matsukura et a1.,15' other studies, including our previous experiment, failed to demonstrate tumor. promoting activity of chemicals in twostage rat glandular stomach tumorigenesiṣ22. 25) In the mouse skin model, a single administration of a very small amount of carcinogen such as 7,12 - dimethylbenz [a] anthracene is sufficient for initiation. In contrast, with the glandular epithelium of the stomach, a relatively long period of administration of a carcinogen is required for initiation. The rat MNNG model for gastric carcinogenesis has provided a suitable system for evaluation of the modifying effects of chemicals on gastric carcinogenesis. Even in this model, however, treatment with MNNG alone for over 12 weeks is necessary for induction of a basal level of preneoplastic lesions. "I On the other hand, when combined with administration of a high concentration of sodium chloride, an 8-week treatment with MNNG results in a low incidence of gastric tumors in rats. Our previous experiment demonstrated a significant modifying effect of sodium chloride on the initiation stage of MNNG-carcinogenesis in rats"' and sodium chloride might act as co-initiator. The present investigation has confirmed our previous observation. It was also demonstrated that subsequent administration of sodium chloride or saccharin increased the incidence of neoplastic and preneoplastic lesions of the glandular stomach in rats. This result suggests a possible promoting effect of sodium chloride or saccharin on MNNG-induced gastric tumors in rats. However, the difference is only significant with respect to preneoplastic hyperplasias in the group receiving sodium chloride. One possible reason is the short duration of the experiment, limiting the potential of the compounds to promote the development of adenocarcinomas. Further confirmation is required regarding the 498 Gano

6 TUMOR PROMOTION IN GASTRIC CARCINOGENESIS promoting effect of saccharin on gastric carcinogenesis. The histological classification of the MNNG-induced lesions in Table I was similar to that in previous studies.25, 26) Although the tumor types occurring in each group appeared to be unrelated to the subsequent treatment with chemicals, the incidence of each lesion was higher in group 2 than group 1; 4 adenocarcinomas (20%) and 8 preneoplastic hyperplasias (40%) were found in the pyloric area in group 2 compared with only 7.7% of adenocarcinomas and 12.8% incidences of preneoplastic hyperplasias in group 1. Since the progression of MNNG-induced lesions from preneoplastic hyperplasias to adenocarcinomas has been well characterized,", 12) the results provide strong evidence for a promoting action of sodium chloride in gastric carcinogenesis. In our experiment, as in several previous experiments using MNNG, tumors of the duodenum also occurred, treatment with MNNG in combination with sodium chloride serving as with gastric carcinogenesis to in creasethe yield of carcinomas. The mechanisms underlying the effect of sodium chloride on gastric carcinogenesis remain unclear. One possibility is that initiation is indirectly enhanced by gastric irritation and damage to the mucous membrane. The gastric mucosa contains several kinds of acid mucopolysaccharides, including hyaluronic acid, the viscosity of which is known to decrease markedly in the presence of sodium chloride. It has earlier been proposed that the mucosal barrier in the glandular stomach is important in preventing the induction of stomach carcinomas in rats27 and sodium chloride might reduce its protective action by allowing easier access of the carcinogen and therefore increased damage to the gastric mucosa to occur. Although the mechanism of action of various organospecific promoters is unclear, one common observation during the promoting stage is the induction of hyperplasia or of increased DNA synthesis in target sites." 5.17 In this study it was also shown that the enhancing treatments with sodium chloride and saccharin induced diffuse proliferative changes in the superficial epithelium or generative zone of the glandular stomach, whereas the nonenhancers, phenobarbital and aspirin, showed neither effect, although focal erosive lesions could be seen in the aspirin groups. Such a proliferation-stimulating mechanism found diffusely in the gastric mucosa could be important in enhancing gastric carcinogenesis by sodium chloride and saccharin. However, elucidation of the exact biochemical alterations underlying the effect of sodium chloride or saccharin on glandular stomach tumors will require further investigation. In this study, phenobarbital did not increase tumor incidence, and aspirin appeared to show an inhibitory activity, although because of the relatively low incidence of gastric tumors in the control group in this experiment, no statistical significance of the effect could be established. However, the tendency for a decrease of gastric tumors observed in group 5 might be expected in relation to the pharmacological properties of aspirin, which acts as a potent inhibitor of prostaglandin synthesis."' In animal studies, prostaglandin synthetase inhibitors, such as indomethacin and aspirin, have been reported to be effective in reducing the rate of tumor growth in a number of gastrointestinal sites. 16, 16, 24) Further studies are needed to evaluate fully the inhibitory effect of aspirin on gastric carcinogenesis. ACKNOWLEDGMENTS We gratefully acknowledge the assistance of Dr. M. A. Moore in the preparation of this manuscript. This work was supported in part by Grants-in-Aid for Cancer Research from the Ministry of Education, Science and Culture (1982, 1983). (Received Feb. 14, 19841Accepted April 26, 1984) 75(6)

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