role of prostaglandins in the early phases of experimental gastric carcinogenesis in the rat

J Cancer Res Clin Oncol (1989) 115:253-258

J~om~lof

Cancer esearch Clinical �9 �9 Springer-Verlag 1989

Role of prostaglandins in the early phases of experimental gastric carcinogenesis in the rat * A. Materia a, G. Silecchia a, E. Spaziani 1, p. Mariani 1, L. Scucchi 2, p. Mingazzini 2, C. Favalli a, E. Garaci a and N. Basso a 1 II Clinica Chirurgica University "La Sapienza", 2 Department of Biopathology University "La Sapienza", 3 Department of Experimental Medicine, University "Tor Vergata", Rome, Italy

Summary. The study was initiated to evaluate the effect of N-methyl-N-nitro-N-nitrosoguanidine (NG) on gastric intraluminal prostaglandin release during a 30-day treatment period and to investigate the effect of a stable prostaglandin E a analogue (misoprostol) on NG-induced gastric mucosal damage during the same time period. Samples of gastric juice (1 h) were obtained from 40 male Sprague-Dawley rats with chronic gastric fistulas, in basal conditions and after 5, 15 and 30 days of continuous oral administration of NG (120 rag/l) or tap water. Aliquots of gastric juice were titrated with 0.1 M NaOH.Other aliquots were extracted with ethyl acetate and subjected to specific radioimmunoassay for prostaglandin E 2. The severity of gastric mucosal lesions was evaluated in 60 rats after 5 days and 30 days of continuous oral administration of NG (120 rag/l) or NG plus misoprostol (200 gg/kg-a/day-1) or tap water, and a histological study was carried out. Administration of NG induced a significant decrease of gastric intraluminal prostaglandin E 2 concentration at 15 and 30 days. Oral administration of misoprostol, at non-antisecretory doses, protected the rats against NG-induced gastric mucosal damage. Prostaglandins may be involved in the early phases of experimental gastric carcinogenesis.

Key words: Gastric experimental carcinogenesis - N- Methyl-N-nitro-N-nitrosoguanidine - Prostaglandins - Prostaglandin analogues - Gastric juice.

* This work was supported by grants from the Ministry of Education (MPI)

Offprint requests to." Alberto Materia, II Clinica Chirurgica, Poli- clinico Umberto I, Universita' degli Studi "La Sapienza', 1-00161 Roma, Italy

Introduction

Several studies have confirmed that N-methyl-N-nitro-N-nitrosoguanidine (NG) is a valuable tool for production of adenocarcinomas in the rat stomach since it is easily dissolved in tap water and adminis- tered orally, and produces lesions in over 70% of rats treated for 10-12 months (Deschner et al. 1979; Sugi- mura and Fujimura 1967; Kunze et al. 1979; Nagai et al. 1980; Ochara et al. 1980; Saito et al. 1970; Sugimura et al. 1970; Sugimura and Kawachi 1973). The sequential morphological changes of NG-induced gastric mucosal damage in the rat are characterized by four types of lesions: erosions, regenerative hyperplasia, adeno- matous hyperplasia and adenocarcinoma (Saito et al. 1970). Previous reports showed that repeated NG action on regenerating cells favors carcinogenesis in the eroded area, suggesting a close relationship between cytotoxic and carcinogenic effects for this compound (Saito et al. 1970; Tabuchi et al. 1974). However, the mechanism of formation of early NG-induced gastric mucosal lesions is not completely understood, nor it is known whether the prevention of mucosal erosions, and consequently of regenerative hyperplasia, could interfere with gastric carcinogenesis. Several studies have emphasized the prominent role played by prostaglandins in the protection of the gastric mucosa against a number of ulcerogenic agents and procedures (Jacobson et al. 1976; Robert 1979; Robert et al. 1979). However, the role of endogenous prostaglandins in experimental gastric carcinogenesis is not known.

This study was initiated to investigate the changes of gastric intraluminal prostaglandins during the early phases (30 days) of NG-induced gastric carcinogenesis in the rat and to evaluate the effects of exogenous prostaglandin administration on NG-induced gastric mucosal lesions during the same period.

254 A. Materia et al.: Prostaglandins and gastric carcinogenesis

Materials and Methods

Animals and chemicals. One hundred Sprague-Dawley male rats, weighing 220 250 g (Charles River, Como, Italy) were utilized in the study. The rats were conditioned to the room environment for at least 15 days prior to the beginning of each experiment. All rats were deprived of food, but not of water, for 24 h prior to surgery or collection of gastric juice and were housed in individual cages with wide wire bottoms to prevent coprophagia. Experiments were performed between 8 a.m. and 2 p.m. 1-Methyl-3-nitro-l-nitrosoguanidine (N N' N) 97% (Aldrich-Chemic, Steinheim, FRG) was used as the carcinogen, diluted with tap water at a concentration of 120 rag/l. The solution was kept in dark bottles to avoid degradation by light. The solution was prepared and changed every other day. The animals were allowed to drink NG solution and to eat commercial rat chow ad libitum.

Experimental design

Study 1. In 40 rats a chronic gastric fistula was created, with a steel cannula (external diameter 12 ram) positioned in the anterior wall of the glandular stomach through a laparotomy under light ether anes- thesia. The cannula was secured to the abdominal wall and dosed with a screw steel plug. One week after surgery, the rats were trained to moderate restraint in Bollman cages for 20-min-periods each day for 2 weeks. The rats were then divided into two groups: group A (20 animals) received NG solution for 30 days; group B (20 animals) received tap water only for 30 days. In all rats gastric juice was col- lected through the gastric fistula for i h on days 0, 5, 15, and 30. After collection, aliquots of gastric juice were titrated with 0.1 M NaOH. Other aliquots were centrifuged at 4 ~ (2500 rpm x 5 min) and 2 ml supernatant was immediately extracted with 10 ml ethyl acetate. After centrifugation, the organic phase was evaporated in a stream of nitrogen and then dissolved in 2 ml phosphate buffer. Prostaglandin E2 concentration was measured by radioimmunoassay using specific antibodies with minimal cross-reactivity with other types of prostaglandins (< 4%). l~5I-labeled prostaglandin E 2 was used as tracer. Antibodies, iodinated prostaglandin E2 and standards were obtained from New England Nuclear, Boston, Mass.

Study 2. Three groups of 20 rats each were studied: group A received NG solution for 30 days; group B received NG plus a stable analogue of prostaglandin E 1 (Dajani et al. 1976) (misoprostol, Searle Chemi- cals, Skokie, Ill, USA) (200 lag/kg- 1 day- 1) dissolved in tap water; group C received tap water only. Ten rats from each group were sac- rificed on day 6 and the remaining ten on day 30. The stomach of each rat was removed, opened along the greater curvature, pinned flat on a cork board and rinsed with saline solution for gross examination. The severity of gastric erosions was evaluated in a blind fashion using a dissecting microscope. Only gastric mucosal lesions occurring in the glandular part of the mucosa were considered. The length and width of each lesion were measured to the nearest 0.2 mm and the surface area of erosion was calculated by assuming each erosion to be an ellipse (length x width/4) and the total area was summed and expressed in mm z (Kaufman and Grossman 1978). The average lesion area for a group of animals was calculated as the mean of the lesion area for each rat +_ the standard error of the mean (SEM). After fixation in 10% neutral formalin, all the glandular stomach and duodenum was cut into 4-ram-wide strips and embebbed in paraffin. Several sections 4 lam thick were obtained from each spec- imen and stained with hematoxylin/eosin, periodic and / Schiff and phosphotungstic and / hematoxylin methods.

Study 3. In a separate experiment, the effect 0fmisoprostol on gastric secretion, at the doses cited above, was investigated in five rats that had chronic gastric fistula. In these animals gastric acid was measured in basal conditions and after 24 h of continuous oral

administration of the prostaglandin analogue at the dose 200 lag/ kg-1 day-1. Statistical evaluation of the data was performed by means of the two-way analysis of variance and by Student's t-test for paired values. P values less than 0.05 were considered as significant.

Results

Water intake was comparable in all rats with an average of 45 ___ 12 ml/day and was consistent throughout the entire experimental period. Food intake was also comparable and consistent in all rats.

Study 1

The mean intake of NG was 6.0 +_ 1.5 rag/day for each rat. The gastric secretion volume and HC1 concentration on days 0, 5, 15, and 30 are summarized in Figs. 1 and 2. No significant changes in either secretion volume or HC1 concentration were found. Prostaglandin E 2 concentration in the gastric juice was shown to be 1105_+98.5 at day 0, 865_+125.8 at day 5, 645_+66.3 at day 15, and 596+_48.3 at day 30 with statistically significant differences (P<0.05) between day 0 and day 15 and day 0 and day 30 (Fig. 3). Consequently,

ml/h 5

al

E -~4 >

= :3

cn 2 ._o

0 0 5 days 15 days 30 days

[ ] control [ ] NG n.s,

Fig. 1. Gastric secretion volume in N-methyl-N-nitro-N-nitrosoguanidine (NG)-treated animals and controls

J~Ea/ml 20 T

15

10 =o

8 U 5 -r

0 [ ] control

5 days 15 days I~NG

30 days

n.s.

Fig. 2. Gastric HC1 concentration in NG-treated animals and controls

A. Materia et al.: Prostaglandins and gastric carcinogenesis

Pg/ml

1200

900

o ~ 600 c o

300

O.

0 0 5 days 1,5 days 30 days

[] Control [] NG = # =p<O,05

Fig. 3. Gastric intraIuminal prostaglandin E 2 concentration in rats treated with NG and in control rats

Z mm

20

255

15 113

m 10 t -

9_ t~ _w 5

6 days

[ ] control [ ] NG

30 days

[ ] NG+PG * # = p<O,05

Fig. 4. Average lesion area (mm 2) on days 6 and 30 in rats treated with NG, NG plus misoprostol and tap water (control)

since the secretion volume did not change over the experimental period, the total output of prostaglandin E 2 paralleled the changes in concentration. No varia- tions in gastric instraluminal prostaglandin Ez were observed in control animals.

Study 2

The mean intake of NG was 6,7 + 2.3 rag/day for each rat; the mean intake of misoprostol was 180 + 25 I-tg/ kg-1 day-1 for each rat. The average lesion area at day 6 was 15.6+1.4mm 2 in NG-treated rats, 1.2___ 0.2 mm 2 in rats treated with NG plus misoprostol (P < 0.05) and 0 in the controls. At day 30, the average lesion area was 9.8 +0.7 mm 2 in NG-treated animals and 0.7 + 0.2 mm 2 in animals treated with NG plus misoprostol (P< 0.05). No mucosal lesions were observed in controls (Fig.4). No animal in either group experienced diarrhea or showed discomfort.

Study 3

Gastric secretion volume, measured in rats with gastric fistulas, was 6,1 _+ 0.4 ml/h in basal conditions and 5.2_+ 0.4 ml/h (P > 0.05) after 24 h of continuous oral administration of misoprostol (200 lag/kg). The HC1 concentration was 18.0 ___ 0.6 gequiv/ml in basal conditions and 16.6+0.04 gequiv/ml after misoprostol administration (P > 0.05).

Histological findings

At day 6, 24 deep erosions were observed in the glandular stomach of rats treated with NG (Fig. 5). Out of the eroded areas, antral mucosa showed degenerative and regenerative features (Fig. 6).

In the rats treated with NG plus misoprostol, no deep erosions were seen, and only 4 superficial erosions were observed. Out of these areas, the mucosa

Fig. 5. Mucosa of the glandular stomach of a rat treated for 5 days with NG. A deep erosion filled with necrotic debris is shown (H&E x 100)

appeared normal (Fig. 7). No significant gastric mucosal lesions were found in control animals.

At the end of the experiment (day 30), 21 deep erosions were found in NG-treated animals, 15 of them showing a pattern of coagulative necrosis. Regenerat- ive hyperplasia, without atypical cells, was present (Fig. 8).

In the animals treated with NG plus misoprostol, only 1 deep erosion of the antrum-duodenal junction and 2 small superficial erosions of the glandular stomach were found (Fig. 9). In these animals, slight areas of epithelial degeneration and mild antral inflamma-


Fig. 6. Antral mucosa of a rat treated with NG for 5 days. Nuclear pyknosis and karyorrhexis are evident. The nuclear enlargement, hy- perchromasia and the high mitotic rate are expressions of regenera- tion (H&E x 400)

Fig. 8, Mucosa of the glandular stomach of a rat, treated for 30 days with NG, showing regenerative hyperplasia of the foveolar epithelium (PAS x 100)

Fig.7. Mucosa of the glandular stomach of a rat treated with NG plus misoprostol for 5 days. No abnormalities of the mucosa are evident except for a slight edema of the lamina prepria (H&E x 400)

Fig. 9. Mucosa of the glandular stomach of a rat, treated for 30 days with NG plus misoprostol. A shallow erosion of foveolar epithelium sparing the a, trat glands is shown (H&E x 400)

A. Materia et al.: Prostaglandins and gastric carcinogenesis 257

tory reaction were observed. Otherwise, gastric mucosa appeared normal and regenerative hyperplasia was a sporadic feature.

Discussion

On the basis of current data there is strong evidence that N-nitroso compounds can be causative factors in the aetiology of human gastric cancer (Mirvish 1983; Read 1986) and NG has been shown to be one of the most valuable agents employed in experimental gastric carcinogenesis (Deschner et al. 1979).

It is generally accepted that the formation of erosions, which represent the first NG-induced gastric mucosal lesions, is followed by regenerative hyperplasia. The possibility that this lesion is an initial step in cancer development has been postulated (Jacobson et al. 1976; Tabuchi et al. 1974).

In the present study we observed, from the 5th day of treatment with NG, erosions mostly located in the body of the stomach. After 30 days, deep erosions with a pattern of coagulative necrosis were present, as- sociated with areas of regenerative hyperplasia. These findings can be related to a direct effect of NG on the surface epithelium. In this study NG did not affect gastric secretion volume or HC1 concentration over the entire experimental period confirming that NG acts directly on gastric epithelial cells. On the other hand, NG significantly decreased the concentration of intraluminal prostaglandin E2, expressing a reduced availability of prostaglandins at the gastric mucosal surface. Prostaglandins have been shown to be ac- tively synthesized by the gastric mucosa and a decreased prostaglandin biosynthesis has been observed in several acute and chronic gastric ulcer models (Basso et al. 1983, 1985; Konturek 1984; Schlegel et al. 1977; Wright et al. 1982) suggesting that endogenous prostaglandins may play a role in the protection of the gastric mucosa against cytotoxic agents. On these bases, it is possible to hypothesize that endogenous prostaglandins are involved in the formation of NG- induced musosal lesions. Whether the prostaglandin inhibition represents a consequence of the extensive gastric mucosal damage or a preliminary effect of NG action contributing to the damage itself it is not known. Furthermore, the administration of cytopro- tective doses of a stable prostaglandin analog significantly prevented the cytotoxic effects of NG, with a marked reduction of mucosal erosions. At day 30, in rats treated with misoprostol the regenerative hyperplasia was a sporadic feature and deep erosions were absent. These results indicate that exogenously admin- istered non-antisecretory doses of prostaglandins are able to antagonize the early changes of the gastric epithelium induced by NG. Since, repeated sequences of

tissue damage and regenerative processes tend to pro- mote cancer de,~elopment, and since exogenous prostaglandins seem to decrease the NG-induced early cytotoxic lesion, an influence of these compounds on the outcome of dysplasia or carcinoma may be hypothe- sized. Long-term studies are underway to investigate the effects of prostaglandins on the development of neoplastic lesions. As for the mechanism of prostaglandin-induced protection on the early gastric mucosal damage by NG, it can be found in the growing list of hypotheses already compiled to explain the protection exerted by these compounds against several ulcerogenic agents and methods (Robert 1979; Robert et al. 1979). In conclusion, the results of this study sug- gest that endogenous prostaglandins may play a role in the early phases of gastric experimental carcinogenesis and that exogenous prostaglandins, at non-antisecretory doses, prevent the formation of early NG-induced gastric mucosal lesions.

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Received 5 December 1988/Accepted 31 January 1989

role of prostaglandins in the early phases of experimental gastric carcinogenesis in the rat

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