Induction of Intestinal Metaplasia in the Stomachs of Rats by N-Methyl-N'-nitro-N-nitrosoguanidine 1,2 Norio Matsukura,
3
Takashj Kawachi,
and Teruyuki Hirota
3
Koji Sasajima,
3
Tomono. Sano,
3
Takashi Sugimura,
3
3,4
ABSTRACT -N-Methyl-N'-nitro-N-nitrosoguanidine (MNNG) was administered orally to male Wistar rats at a concentration of 83 ILg/ml In the drinking water for 2, 4, 5, and 7 months; the rats were killed at about month 15. Intestinal metaplasia was found in the stomachs of 80-100% of the rats treated with MNNG for 4 or more months, of 37.5% treated with MNNG for 2 months, and of 10% of the controls. Metaplastic glands, composed of goblet cells and columnar cells with striated borders, were found in the pyloric region. Paneth's cells were found at the bottom of metaplastic glands in a rat treated with MNNG for 4 months. The incidence of well-differentiated adenocarcinomas of the stomach was 63-90% in rats treated with MNNG for 4 or more months and 25% in those treated with MNNG for 2 months.-J Natl Cancer Inst 61: 141-144, 1978.
Epidemiologically, histologically, and biochemically, intestinal metaplasia is closely related to well-differentiated adenocarcinoma of the human stomach (1-5). Sugimura and Fujimura (6) first induced stomach carcinomas in rats by treatment with MNNG. Since then, stomach carcinomas have been studied extensively in animals (7, 8), but the development of intestinal metaplasia during the process of gastrocarcinogenesis has not been reponed. Attempts have been made to induce intestinal metaplasia by X-ray irradiation (9) or by injection of 3-methycholanthrene into the submucosa of the stomach (10), but success has been limited. In this work, we studied the induction of intestinal metaplasia in rats after oral administration of MNNG for various periods.
MATERIALS AND METHODS Male Wistar rats (Nihon Rat Co., Ltd., Saitama, japan), approximately 170 g each, were used. MNNG (Aldrich Chemical Co., Inc., Milwaukee, Wis.) was dissolved in deionized water at a concentration of 1.0 mg/ml as stock solution. The stock solution was diluted to 83 /-Lg/ml with tap water just before it was given to rats ad libitum. Of the 5 groups studied (IS rats/group), groups I, II, III, and IV received MNNG for 2, 4, 5, and 7 months, respectively, and then normal tap water; the MNNG intakes per rat in groups I, II, III, and IV were estimated to be ISO, 300, 375, and 525 mg, respectively. The rats in group V received tap water throughout the experiment. All groups were maintained on basic rat chow (CE-2; CLEA japan Inc., Tokyo, japan). Most of the rats were killed about IS months from the beginning of the experiment. The stomach was opened along the greater curvature, pinned flat on a cork mat, and fixed with 10% neutralized Formalin solution. The fixed stomach was then cut into 2-mm-wide, longitudinal strips. The strips were routinely stained with H & E, VoL. 61. NO. I. JULY 1978 Downloaded from https://academic.oup.com/jnci/article-abstract/61/1/141/917683 by Durham University user on 06 March 2018
and some strips were stained with Alcian blue-PAS. Careful histologic examination was done and the number of metaplastic glands in all the sections of each glandular stomach was ascertained.
RESULTS Animals in the 4 groups treated with MNNG gained body weight to the same extent as the controls. In groups I, II, III, IV, and V,S, 5, 4, 5, and 3 rats, respectively, died of pneumonia by experimental day 285; no intestinal metaplasia or tumors were detected in these rats. Morever, 2, 2, I, 2, and 2 rats, respectively, which died of pneumonia between day 350 and day 452, could not be examined histologically because of autolysis of their stomachs. Intestinal metaplasia and adenoma of the stomach were first found on day 341 in a rat in group II. Metaplastic glands consisted of goblet cells and nonmucus-secreting columnar cells with striated borders (fig. I). The goblet cells in metaplastic glands were stained with Alcian blue, like those in duodenal glands (fig. 3). Paneth's cells with acidophilic granules were found at the bottom of metaplastic glands (fig. 2) in a specimen from a rat in group II but were not found in rats of other groups. Metaplastic glands were hyperplastic, showing more mitoses than did normal pyloric glands. Metaplastic glands were most frequent in foci among pyloric glands in the proximal pyloric region. They were also present in the distal pyloric region, but in lower incidence, and were not found in the fundic region. They were not in contact with erosions or regenerative epithelium and were not always located near adenomas or adenocarcinomas. Data on the incidences of intestinal metaplasia and tumors in each group are shown in table 1. Metaplastic glands were found in 3 of 8 rats in group I and in 8 of 8 rats in group II. The incidences of intestinal metaplasia A88REVIATIONS USED: H & E=hematoxylin and eosin; MNNG=Nmethy I-N'-nitro-N -nitrosoguanidine; P AS=periodic acid-Schiff.
Received Augusl 31. 1977; accepted February 15, 1978. Supported by Grants-in-Aid for Cancer Research from the Ministry of Education, Science, and Culture, the Ministry of Health and Welfare, the Princess Takamatsu Cancer Research Fund. and the Society for Promotion of Cancer Research. Japan. 3 National Cancer Center Research Institute, Tsukiji, Chuo-ku. Tokyo 104. Japan. 4 We thank Dr. Masayuki Itabashi, National Cancer Center Research Institute. for his helpful suggestions. I
2
141
j
NATL CANCER INST
142 Matsukura, Kawachi, Sasajima, et at TABLE I.-Incidence of intestinal metaplasia and tumors in the
stomachs of rats given MNNG
Period of MNNG Group adminisNo. tration. mo I
2
II
4
III
5
IV
7
V
None (controll
Experimental period. day' 441 441 441 442 442 442 442 442 341 437 437 437 437 437 440 440 441 441 441 441 441 441 441 520 520 520 400* 419* 435 437 437 438 459 461 412* 443 443 443 443 443 450 450 450 450
No. of metaplastic glands b
Tumor in the glandular stomach
3 0 0 0 0 0 6 14 2 9 3 8 1 2 1 2 7 21 4 0 1 4 0 1 2 1 8 2 3 2 3 3 6 2 0 0 0 0 2
None None Adenoma Adenocarcinoma Adenoma Adenoma Adenocarcinoma Adenoma Adenoma Adenocarcinoma None Adenocarcinoma Adenocarcinoma Adenocarcinoma Adenoma Adenocarcinoma Adenocarcinoma Adenocarcinoma Adenocarcinoma Adenocarcinoma Adenoma Adenocarcinoma Adenocarcinoma Adenocarcinoma Adenocarcinoma Adenocarcinoma None Sarcoma Adenocarcinoma Adenocarcinoma Adenoma Adenocarcinoma Adenocarcinoma Adenocarcinoma None None None None None None None None None None
0
0 0 0 0
• From day 1. Asterisk indicates natural death of animal. All other rats were killed on day indicated. b In all the 2-mm-wide. longitudinal strips from a single glandular stomach.
in groups I and II were significanlly different as determint'd by Student's t-test (P
3
Takashj Kawachi,
and Teruyuki Hirota
3
Koji Sasajima,
3
Tomono. Sano,
3
Takashi Sugimura,
3
3,4
ABSTRACT -N-Methyl-N'-nitro-N-nitrosoguanidine (MNNG) was administered orally to male Wistar rats at a concentration of 83 ILg/ml In the drinking water for 2, 4, 5, and 7 months; the rats were killed at about month 15. Intestinal metaplasia was found in the stomachs of 80-100% of the rats treated with MNNG for 4 or more months, of 37.5% treated with MNNG for 2 months, and of 10% of the controls. Metaplastic glands, composed of goblet cells and columnar cells with striated borders, were found in the pyloric region. Paneth's cells were found at the bottom of metaplastic glands in a rat treated with MNNG for 4 months. The incidence of well-differentiated adenocarcinomas of the stomach was 63-90% in rats treated with MNNG for 4 or more months and 25% in those treated with MNNG for 2 months.-J Natl Cancer Inst 61: 141-144, 1978.
Epidemiologically, histologically, and biochemically, intestinal metaplasia is closely related to well-differentiated adenocarcinoma of the human stomach (1-5). Sugimura and Fujimura (6) first induced stomach carcinomas in rats by treatment with MNNG. Since then, stomach carcinomas have been studied extensively in animals (7, 8), but the development of intestinal metaplasia during the process of gastrocarcinogenesis has not been reponed. Attempts have been made to induce intestinal metaplasia by X-ray irradiation (9) or by injection of 3-methycholanthrene into the submucosa of the stomach (10), but success has been limited. In this work, we studied the induction of intestinal metaplasia in rats after oral administration of MNNG for various periods.
MATERIALS AND METHODS Male Wistar rats (Nihon Rat Co., Ltd., Saitama, japan), approximately 170 g each, were used. MNNG (Aldrich Chemical Co., Inc., Milwaukee, Wis.) was dissolved in deionized water at a concentration of 1.0 mg/ml as stock solution. The stock solution was diluted to 83 /-Lg/ml with tap water just before it was given to rats ad libitum. Of the 5 groups studied (IS rats/group), groups I, II, III, and IV received MNNG for 2, 4, 5, and 7 months, respectively, and then normal tap water; the MNNG intakes per rat in groups I, II, III, and IV were estimated to be ISO, 300, 375, and 525 mg, respectively. The rats in group V received tap water throughout the experiment. All groups were maintained on basic rat chow (CE-2; CLEA japan Inc., Tokyo, japan). Most of the rats were killed about IS months from the beginning of the experiment. The stomach was opened along the greater curvature, pinned flat on a cork mat, and fixed with 10% neutralized Formalin solution. The fixed stomach was then cut into 2-mm-wide, longitudinal strips. The strips were routinely stained with H & E, VoL. 61. NO. I. JULY 1978 Downloaded from https://academic.oup.com/jnci/article-abstract/61/1/141/917683 by Durham University user on 06 March 2018
and some strips were stained with Alcian blue-PAS. Careful histologic examination was done and the number of metaplastic glands in all the sections of each glandular stomach was ascertained.
RESULTS Animals in the 4 groups treated with MNNG gained body weight to the same extent as the controls. In groups I, II, III, IV, and V,S, 5, 4, 5, and 3 rats, respectively, died of pneumonia by experimental day 285; no intestinal metaplasia or tumors were detected in these rats. Morever, 2, 2, I, 2, and 2 rats, respectively, which died of pneumonia between day 350 and day 452, could not be examined histologically because of autolysis of their stomachs. Intestinal metaplasia and adenoma of the stomach were first found on day 341 in a rat in group II. Metaplastic glands consisted of goblet cells and nonmucus-secreting columnar cells with striated borders (fig. I). The goblet cells in metaplastic glands were stained with Alcian blue, like those in duodenal glands (fig. 3). Paneth's cells with acidophilic granules were found at the bottom of metaplastic glands (fig. 2) in a specimen from a rat in group II but were not found in rats of other groups. Metaplastic glands were hyperplastic, showing more mitoses than did normal pyloric glands. Metaplastic glands were most frequent in foci among pyloric glands in the proximal pyloric region. They were also present in the distal pyloric region, but in lower incidence, and were not found in the fundic region. They were not in contact with erosions or regenerative epithelium and were not always located near adenomas or adenocarcinomas. Data on the incidences of intestinal metaplasia and tumors in each group are shown in table 1. Metaplastic glands were found in 3 of 8 rats in group I and in 8 of 8 rats in group II. The incidences of intestinal metaplasia A88REVIATIONS USED: H & E=hematoxylin and eosin; MNNG=Nmethy I-N'-nitro-N -nitrosoguanidine; P AS=periodic acid-Schiff.
Received Augusl 31. 1977; accepted February 15, 1978. Supported by Grants-in-Aid for Cancer Research from the Ministry of Education, Science, and Culture, the Ministry of Health and Welfare, the Princess Takamatsu Cancer Research Fund. and the Society for Promotion of Cancer Research. Japan. 3 National Cancer Center Research Institute, Tsukiji, Chuo-ku. Tokyo 104. Japan. 4 We thank Dr. Masayuki Itabashi, National Cancer Center Research Institute. for his helpful suggestions. I
2
141
j
NATL CANCER INST
142 Matsukura, Kawachi, Sasajima, et at TABLE I.-Incidence of intestinal metaplasia and tumors in the
stomachs of rats given MNNG
Period of MNNG Group adminisNo. tration. mo I
2
II
4
III
5
IV
7
V
None (controll
Experimental period. day' 441 441 441 442 442 442 442 442 341 437 437 437 437 437 440 440 441 441 441 441 441 441 441 520 520 520 400* 419* 435 437 437 438 459 461 412* 443 443 443 443 443 450 450 450 450
No. of metaplastic glands b
Tumor in the glandular stomach
3 0 0 0 0 0 6 14 2 9 3 8 1 2 1 2 7 21 4 0 1 4 0 1 2 1 8 2 3 2 3 3 6 2 0 0 0 0 2
None None Adenoma Adenocarcinoma Adenoma Adenoma Adenocarcinoma Adenoma Adenoma Adenocarcinoma None Adenocarcinoma Adenocarcinoma Adenocarcinoma Adenoma Adenocarcinoma Adenocarcinoma Adenocarcinoma Adenocarcinoma Adenocarcinoma Adenoma Adenocarcinoma Adenocarcinoma Adenocarcinoma Adenocarcinoma Adenocarcinoma None Sarcoma Adenocarcinoma Adenocarcinoma Adenoma Adenocarcinoma Adenocarcinoma Adenocarcinoma None None None None None None None None None None
0
0 0 0 0
• From day 1. Asterisk indicates natural death of animal. All other rats were killed on day indicated. b In all the 2-mm-wide. longitudinal strips from a single glandular stomach.
in groups I and II were significanlly different as determint'd by Student's t-test (P
