[ Gann, 75, 1053-1057; December, 1984]
STUDY OF THE PROMOTING EFFECT OF SODIUM CHLORIDE ON GASTRIC
CARCINOGENESIS BY N-METHYL-N'-NITRO-N-NITROSOGUANIDINE IN
INBRED WISTAR RATS
Hiroko OHGAKI,*1 Tamami KATO,*1 Kazuhide MORINO,*1 Norio MATSUKURA,*1 Shigeaki SATO,*1 Shozo TAKAYAMA*2 and Takashi SUGIMURA*1
*1 Biochemistry Division, National Cancer Center Research Institute, 1-1, Tsukiji 5 chome, Chuo-ku, Tokyo 104 and *2 Department of Experimental Pathology, Cancer Institute, 37-1, Kami-Ikebukuro 1 chome, Toshima-ku, Tokyo 170
The effect of sodium chloride on the promotion stage of gastric carcinogenesis by N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) was studied in male inbred Wistar rats. Rats in group I were given MNNG at a concentration of 50ƒÊg/ml in their drink ingwater for 12 weeks and then 1ml of saturated NaCl solution intragastrically once a week until experimental week 65. Rats in group II were given MNNG for 12 weeks and then 1ml of distilled water intragastrically once a week until week 65. Rats in group III were not treated for the first 12 weeks and were then given 1ml of saturated NaCl solution intragastrically once a week until week 65. The incidence of adenoma toushyperplasias in the glandular stomach was significantly higher in group I than in group II, but the incidences of gastric adenocarcinomas and adenomas in groups I and II were not significantly different. No neoplastic or preneoplastic changes were observed in the stomach in group III.
Key words: Sodium chloride-Promoter-Gastric carcinogenesis-MNNG-Rat
Gastric cancer is still the most frequent
neoplasm
in Japan.13) It has been shown
that
the incidence of gastric cancer is higher
in first- than in second-generation Japanese
in Hawaii,3)
and the high incidence of gas
triccancer has been thought to be due
to environmental factors. Epidemiological
studies
have shown that excess intake of
highly
salted foods is closely associated with
gastric
cancer.4,5,12)
Some studies have been made on the ef
fectsof sodium chloride on experimental
gastric carcinogenesis in rats. Tatematsu
et at.18)
demonstrated that intragastric ad
ministrationof saturated NaCl solution si
multaneouslywith carcinogens increased
the incidences
of tumors of the forestomach
induced by 4-nitroquinoline 1-oxide and
of the glandular stomach induced
by
N-methyl-N'-nitro-N-nitrosoguanidine
(MNNG).
Takahashi et al.17) also showed
that 10% NaCl in the diet given concomi
tantlywith MNNG increased both the in
cidenceand
the size of gastric tumors . Shirai
et al.15) reported that administration of 1ml
of saturated NaCl solution before a single
dose
of MNNG increased the incidence of
tumors in the glandular stomach. These re
sultsindicate that NaCl acts as a cocarcinogen
in the initiation stage of gastric carcinogene
sisby MNNG in rats. Possible mechanisms
for the enhancing effects of NaCl on gastric
carcinogenesis are considered to be distur
banceof
the mucous barrier, resulting in
increased penetration of carcinogens to the
gastric mucosa, and/or increased cell pro
liferationdue to repeated injury by NaCl.
It is also important to clarify whether NaCl
is effective in the promotion stage of gastric
carcinogenesis. Several attempts to demon
stratethe
promotive effect of NaCl on car
cinogenesisin
the glandular stomachs of
rats have not been successful.15,17,18)
How
ever,Takahashi
et al.16) recently reported
that administration of 10% NaCl diet in
the promotion stage enhanced the incidence
of preneoplastic hyperplasia in rats that had
been given 100mg/liter
MNNG in their
drinking water plus 10% NaCl diet for 8
weeks in the initiation stage of gastric car
cinogenesis.This result suggested that NaCl
also plays a role in the promotion stage of
gastric carcinogenesis by MNNG. Further
more,Shirai et al.14) found that 5% sodium
H. OHGAKI, ET AL. chloride diet enhanced the incidence of
forestomach papillomas in rats when given as a promoter after a single intragastric application of MNNG.
The present work was undertaken to clarify whether NaCl acts as a promoter in gastric carcinogenesis by MNNG in inbred Wistar strain rats in which lesions related to gastric carcinogenesis by MNNG appear at a specific site of the glandular stomach.
MATERIALS AND METHODS
Male 5-week-old inbred WBN/kob rats, which were purchased from Ishikawa Laboratory Ani mals,Fukaya, Saitama, when they were 4 weeks old, were used. Localized sequential morphological changes of gastric carcinogenesis induced by MNNG, namely, erosions, regenerating epithe lium,adenomatous changes and early and invasive carcinomas have been observed at the midpoint of the lesser curvature of the glandular stomach in this strain of rats.6,7)
One hundred rats were divided into 3 groups. Group I: Forty rats were given 50ƒÊg/ml MNNG in their drinking water for 12 weeks and then 1 ml of saturated NaCl solution by gastric intuba tiononce a week until the end of experimental week 65. Group II: Forty rats were given 50ƒÊg/ml MNNG in their drinking water for 12 weeks and then 1ml of distilled water by gastric intubation once a week until the end of the experiment. Group III: Twenty rats were not treated for the first 12 weeks and then given 1ml of saturated NaCl solu tionby gastric intubation once a week until the end of the experiment.
MNNG was purchased from Aldrich Chemical Co., Inc., Milwaukee, Wisconsin. It was dis solvedin deionized water at a concentration of 500ƒÊg/ml and kept in the dark as a stock solution. The stock solution was diluted 10-fold with tap water just before use and given to rats ad libitum. Rats were housed 5 to a cage and maintained on basal diet (CE-2; CLEA Japan, Inc., Tokyo). Rats were examined every day and were weighed
once a month. Intakes of MNNG solution were measured three times a week.
All animals were carefully autopsied when they died or after being killed because they became moribund or at the end of the experiment
. The stomach was opened along the greater curvature
, pinned flat on a cork board and fixed with 15% neutralized formalin. The fixed stomach was cut along the lesser curvature into strips (2-3mm wide), embedded in paraffin, and then cut into 3ƒÊm-thick sections. The intestine was opened longitudinally and carefully examined macro scopically,and any lesions detected were sectioned . All sections were stained with hematoxylin and eosin.
Histologically, epithelial lesions of the glandular stomach were classified into three types, adeno matoushyperplasias, adenomas and adenocar cinomas,which were in principle based on the reports by Matsukura et al.10) and Takahashi et al.16) Adenomatous hyperplasias were defined as glandular proliferations with structural atypism in the mucosa, which corresponds to preneoplastic hyperplasia as defined by Takahashi et al.15) Ade nomaswere defined as glandular proliferation with structural atypism and downward growth to the submucosa. Adenocarcinomas were defined as excessive glandular proliferations with pronounced structural and cellular atypism invading the sub mucosa,the muscularis propria or the serosa.
Differences in the incidences of lesions were an alyzedby means of the ƒÔ2 test.
RESULTS
The body weights and survival times of the three groups were similar. In groups I and II the average daily intakes of MNNG solution per rat were 16.5ml and 16.2ml, and the average total MNNG consumptions per rat were 74.1mg and 72.7mg, respective ly.The first intestinal adenocarcinoma was found in a rat in group II on day 227. Thus, effective animals were defined as those sur vivingfor more than 227 days.
Table I. Effect of Intragastric Application of Saturated NaCl Solution on the Promoting Stage of Gastric Carcinogenesis Initiated by MNNG in WBN/kob Rats
a) Numbers in brackets show lesions located in the fundic region. b) Significantly higher (P<0.03) than in group II.
EFFECT OF SALT ON GASTROCARCINOGENESIS
The incidences of adenomatous hyper-plasias of the stomach in groups I and II were 23% and 3%, respectively, the differ encebeing significant (P<0.03, Table I). Four adenomatous hyperplasias in group I and one adenomatous hyperplasia in group II were located at the midpoint of the lesser curvature of the pyloric region, but five ade nomatoushyperplasias in group I were in the fundic region (Fig. 1).
The incidences of epithelial tumors in the stomach (adenocarcinomas and ade nomas)in groups I and II were 29% and 11%, respectively, the difference not being significant (0.09<P<0.10). All adenomas and adenocarcinomas in groups I and II were located at the midpoint of the lesser curvature of the pyloric region except for one adenocarcinoma in a rat in group I, which was located at the fundic region con tiguouswith the limiting ridge (Fig. 2). Sarcomas were found in 1 of 35 (3%) in group I and 2 of 38 (5%) in group II, the difference in incidences not being signifi cant.No neoplastic or preneoplastic changes were found in the stomach in group III.
Focal erosion in the fundic and pyloric regions, irregular arrangement of pyloric glands, intestinal metaplasia of the pyloric region, pyloric metaplasia and cystic dila tationof the fundic region were all found in a few rats of groups I and II. Focal erosions in the fundic and pyloric regions, pyloric metaplasia and cystic dilatation of the fundic region were also found in a small number of
rats of group III. No significant difference was found in the incidences of these lesions in different groups.
Intestinal tumors, adenocarcinomas and sarcomas were induced in 4 of 35 rats (11%) in group I and in 4 of 38 rats (11%) in group II. The difference in the incidences of in testinaltumors in groups I and II was not significant. No intestinal tumors were found in group III.
DISCUSSION
The present experiment showed that the incidence of adenomatous hyperplasia in the glandular stomach was higher in rats given NaCl after MNNG treatment than in those given MNNG alone. Since adeno matoushyperplasia is considered to be pre neoplasticin gastric carcinogenesis in rats,16) the results in the present study suggest that NaCl promotes gastric carcinogenesis induced by MNNG.
Moreover, in the group given MNNG and then NaCl, five adenomatous hyper-plasias and one adenocarcinoma were ob servedin the fundic region, which is not the site of tumor induction by MNNG in this strain. When MNNG was given to inbred WBN/kob rats, erosions, regenerating epi thelium,adenomatous changes and car cinomaswere all observed at exactly the same site, the midpoint of the lesser curva tureof the pyloric region.6,7) In fact, all the lesions in group II were located at the
mid-Fig. 1. Adenomatous hyperplasia in the fundic region of the glandular stomach of a rat in group I . Proliferation of epithelium with structural atypism was observed in the fundic region . Hematoxylin and eosin stain . •~30.
H. OHGAKI, ET AL.
point of the lesser curvature of the pyloric region. Thus it is possible that the five ade nomatoushyperplasias and one gastric ade nocarcinomalocated in the fundic region in group I were due to the promoting effect of NaCl.
An increased incidence of preneoplastic hyperplasia has also been observed when 10% NaCl diet is given to rats in the pro motionstage of gastric carcinogenesis ini tiatedby MNNG, although in this experi mentNaCl diet was also given simultaneous lywith MNNG in the initiation stage.16)
These results, together with our findings, suggest that NaCl may have a promoting action on gastric carcinogenesis.
However, these results did not provide conclusive evidence of the promoting effect of NaCl because there was no significant increase in the incidence of gastric tumors. Further experiments with larger numbers of rats and a longer period of exposure to NaCl or more frequent administration of NaCl may more clearly demonstrate the promoting effect of NaCl on gastric carci nogenesisin rats.
Fig. 2. Gastric adenocarcinoma observed in the fundic region contiguous with the limiting ridge in a rat in group I. Hematoxylin and eosin stain. Low magnification. •~8 (A). Higher magnification. •~150 (B).
EFFECT OF SALT ON GASTROCARCINOGENESIS
Recently, NaCl has been shown to induce
ornithine decarboxylase activity and semi
conservativeDNA replication in the gastric
mucosa of rats, suggesting promotion activity
of this chemical in the stomach.2)
Besides the promoting effect of NaCl,
other factors that modify the incidence of
gastric cancers should also be studied. For
instance, sodium taurocholate has recently
been shown to have an enhancing or pro
motingeffect on gastrocarcinogenesis in
rats.1,8,9,11) Such information should be very
useful in considering the causative agents
and prevention of human gastric cancer.
(Received Aug. 1, 1984/Accepted Oct. 31, 1984)
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