Int. J. Cuncer: 52,924-927 (1992) 'L 1992 Wiley-Liss, Inc.
%b :
Publication of the International Union Against Cancer Publication de I Union Internationale Contre le Cancer
PROTECTION BY MUSCIMOL AGAINST GASTRIC CARCINOGENESIS INDUCED BY N-METHYL-" -NITRO-N-NITROSOGUANIDINE IN SPONTANEOUSLY HYPERTENSIVE RATS Masaharu TATSUTA' ', Hiroyasu I I S H I ~Miyako , BABA',Hiroyuki UEHARA', Akihiko NAKAIZUMI' and Haruo T A N I G U C H I ~ Departments of 'Gastrointestiiial Oncology and ?Pathology, The Center for Adult Disease, Obaka, 3-3, Nakumichi I-chome, Higash~nan-kir,Osaka 537, Japan. The effects of prolongedadministration of the gamma-aminobutyric acid receptor agonist muscimol on enhanced induction of gastric carcinogenesis by N-methyl-N'-nitro-N-nitrosoguanid ine (MNNG) in spontaneously hypertensive rats (SHR), and on the norepinephrine concentration in the gastric wall and the labeling index of gastric mucosa were investigated. SHR and normotensive Wistar Kyoto (WKY) rats as controls were given a solution of MNNG (25 pg/ml) for 25 weeks and then i.p. injections of 0.5 mg/kg body weight of muscimol every other day. In control WKY rats, gastric cancers were found in I (7%) of 14 rats examined a t week 52. In SHR treated with NaCl solution only, the incidence of gastric cancers was significantly increased to 50% compared with that in control WKY rats. However, treatment of SHR with muscimol significantly increased its incidence to 12% compared with the value in SHR treated with NaCl solution only. The norepinephrine concentration in the gastric wall and the labeling index of the gastric mucosa were significantly greater in SHR than in WKY rats. Prolonged administration of muscimol to SHR significantly reduced the norepinephrine concentration in the antral portion of the gastric wall or the labeling index of the antral epithelial cells. These findings indicate that long-term treatment of SHR with muscimol attenuated the enhancement of gastric carcinogenesis in SHR.
o 1992 Wilq-Liss, Inc. We have found that, at week 52, the incidence and number of gastric cancers induced by MNNG were significantly greater in SHR than in WKY rats. and that the norepinephrinc concentration of the gastric wall was also higher in SHR (Tatsuta et a/., 1989b). As the norepinephrine concentration is considered to be a marker of the sympathetic nervous system, this tinding suggested that an increase in sympathetic nervous system activity in SHR may enhance the process of gastric carcinogenesis. Unger et 01. (1984) found that injection of the potent GABA agonist muscimol into the lateral brain ventricle lowered the mean arterial blood pressure dose-dependently in SHR, and suggested that this was due to selective suppression of sympathoadrenal activity. Their results indicated that administration of muscimol might attenuate gastric carcinogenesis in SHR, and therefore, in the present work, we examined its effect on gastric canccr induced by MNNG in SHR.
MATERIAL AND METHODS
Animal5 Six-week-old male SHR and age-matched male WKY rats were obtaincd from Hoshino (Tochigi, Japan). SHR were from the colony of the Department of Pharmacology, Jichi Medical School, Tochigi, Japan. In this study, WKY rats were used as controls for SHR. SHR were originally derived from WKY rats and thus these 2 strains had the same genetic origin but not the same hypersensitivity. The rats were housed in suspended, wire-bottomcd metal cages in animal quarters at controlled temperature (21-22°C) and humidity (30-50%), with a 12-hr light, 12-hr dark cycle. and were given free access to regular chow pellets (Oriental Yeast, Tokyo, Japan).
Experimental design Rats of the SHR and WKY strains were given drinking water containing MNNG (25 pg/ml; Aldrich, Milwaukee, WI) for 25 weeks. MNNG was dissolved in deionized water at a concentration of 2 mgikg and kept in a cool, dark place and the stock solution was diluted to 25 pgiml with tap water just before use. Each rat was given 40 ml of MNNG solution (less than a single rat can consume in 48 hr) from a bottle covered with aluminum foil to prevent photolysis of MNNG, and the solution was replenished every other day. At week 26, the WKY and SHR rats were each randomly divided into 2groups, and treated as follows until the end of the experiment at week 52. One group of WKY rats (Group 1: 25 rats) and one group of SHR (Group 3: 30 rats) were given i.p. injections of 2 ml/kg body weight of 0.9% NaCl solution every other day. The other groups of WKY rats (Group 2; 25 rats) and SHR (Group 4; 30 rats) were given i.p. injections of 0.5 mgikg body weight of muscimol every other day. Muscimol (Sigma, St. Louis, MO) in 0.9% NaCl solution was injected in a volume of 2 mlikg body weight. Injections were given between 2 and 3 p.m.
Histological observations Ten rats each from each group were killed at week 30 for measurement of the catecholamine concentration of the gastric wall and the labcling index of the gastric mucosa. Animals that survived for more than 50 weeks were included in effective numbers because the first tumor of the glandular stomach was found in a rat in Group 3 that died at week 50. All surviving animals were killed at the end of the experiment at week 52 and autopsied. The stomach and other organs were carefully examined. The stomach was opened along the greater curvature, pinned flat on a cork mat, and fixed with Zamboni's solution for histological examination. The fixed stomach was cut into longitudinal strips, 3 mm wide. The specimens were then embedded in paraftin, and serial sections 5 pm thick were stained with hcmatoxylin and eosin. Sections were examincd without knowledge of which group they were from. Classification qf gastric cancers Histologically, adenocarcinomas were defined as lesions in which neoplastic cells had penetrated the muscularis mueosae to involve the submucosa or deeper layers. Adenocarcinomas were classified as very well-differentiated, well-differentiated, and poorly differentiated types. Meusurement of cutecholamivies in the gastric wall The epinephrine and NE concentrations in the tissues of the gastric wall were determined by high-performance liquid chromatography as reported previously (Tatsuta et al., 19896) 3Towhom correspondence and reprint requests should be sent A hbreviations: MNNG, N-methyl-N'-nitro-N-nitrosoguanidine, BrdU,
hromodeoxyuridine;NE, norepinephrine;muscimol, 5-arninomethyl-3hydroxyisoxazole. ~~
Received: May 18, 1992 and in revised form August 11, 1992
925
GASTRIC CANCER INHIBITION BY MUSCIMOL IN SHR
at weeks 30 and 52. For this purpose, 5 rats in each group were starved for 12 hr and then given 2 mlikg of 0.9% NaCl solution (Groups 1 and 3) or 0.5 mgikg of muscimol (Groups 2 and 4). Two hours later, the rats were killed by cervical dislocation. Samples of about 50 mg of the wall of the fundic and antral portions o f the stomach were removed from each rat, homogenized with 4 ml of 0.4 N perchloric acid, and centrifuged at 1,lOOg for 10 min. The supernatant was mixed with 1 ml of 0.2 M disodium ethylenediaminetetraacetate (EDTA), and the mixture was adjusted to p H 6.0 with ammonium hydroxide. Then the mixture was added to 300 mg of purified aluminum (Woelm Neutral Active Grade I), and the pH was adjusted to 8.4-8.8 with ammonium hydroxide. The mixture was stirred for 5 min and centrifuged at 10,000 g for 10 min, then the supernatant was aspirated and discarded. The precipitated aluminum was washed twice with distilled water and then shaken vigorously with 2.5 ml of 0.4 N acetic acid. The mixture was centrifuged, the clear supernatant was transferred to a small glass tube and lyophilized for 3 hr, and the residue was dissolved in 0.5 ml of 0.2 N acetic acid. A 50-)*I aliquot of this solution was injected into a liquid chromatographic column (Hitachi 301 I-C gel column, 2.6 X 250 mm). Materials were eluted with 0.1 M KHzPOI containing 0.05% HIPOj at a constant flow rate of 0.5 mlimin at 45.0 ? 0.2”C. The eflluent was mixcd with the reagent for the trihydroxyindol reaction, consisting of 0.0075% potassium ferricyanide, 0.1% ascorbic acid, and 5 N sodium hydroxide. The resulting fluorescent products were measured with a highly sensitive spectrofluorophotometer.
Measrircwient of labeling index of gastric mucosa The labeling index of the gastric mucosa was examined at weeks 30 and 52. The labeling index was measured with an immunohistochemical analysis kit (Becton-Dickinson, Mountain View, CA) for assaying BUdR incorporation (Morstyn et ul.. 1983). For this purpose, 5 rats in each group were starved for 12 hr and then treated i.p. with either 2 mlikg of 0.9% NaCl solution (Groups 1 and 3) or 0.5 mgikg of muscimol (Groups 2 and 4). One hour later, BUdR (20 mgikg) was injected i.p., and after another hour the animals were killed with ether. The stomach was removed and fixed in 70% ethanol for 4 hr, and then cmbcdded in paraffin. Thin sections 3 km thick were immersed in 2 N HCI solution for 30 min and then in 0.1 M Na2B407.Slides were also immersed on 0.3% H20,in methanol for 30 min to block endogenous peroxidase activity, and then treated with 10%’ horse serum. Specimens were incubated with anti-BUdR monoclonal antibody (diluted 1:20) for 2 hr, washed, and stained first with biotin-conjugated horse antimouse antibody (Vector, Burlingame, CA: diluted 1:200) for 30 niin. and then with avidin-biotin-peroxidase complex (Vector) for 30 min. The reaction product was detected with 3,3’-dianiinobcnzidine tetrahydrochloride. Cells that contained BUdR were identified by the presence of a dark pigment over their nucleus. To determine the labeling index of
the gastric mucosa, the numbers of BUdR-labeled and -unlabeled cells in the zone of proliferating cells were counted (Eastwood and Quimby, 1983) without knowledge of which treatment group the samples were from. The zone of proliferating cells in the fundic mucosa was defined as a 250-pm rectangular area between the highest and lowest cells in a well-oriented section. Ten such rectangular areas of each rat were examined. In the antral mucosa, all cells below the highest labeled cell in each pit-gland column were regarded as being within the zone of proliferating cells, and 100 welloriented pit-gland columns in each rat were examined. On the basis of these measurements, we calculated the labeling index as the number of BUdR-labeled cellsitotal number of cells within the proliferating zone.
Statistical analysis Results were analyzed by the Chi-square test or Fisher’s exact probability test, or by one-way analysis of variance with Dunn’s multiple comparison (Miller, 1966). Data are shown as means 5 SE. “Significant” indicates a calculated p-value of less than 0.05. RESULTS
Incidence and histological ppes of gastric cancer One rat in Group 1 died before week 35. No tumors were found in this animal, which was excluded from the effective number. At week 52, the body weights of the SHR group without muscimol treatment were significantly lower than those of the other groups. The incidence and histological type of gastric cancers in MNNG-treated rats are summarized in Table I . In control Group I (WKY), gastric cancers were found in only one (7%) of 14 rats examined. In Group 3 (SHR), the incidence of gastric cancers was significantly higher than in Group 1. Treatment of SHR with muscimol resulted in a significant decrease in the incidence of gastric cancers (Group 4 vs. Group 3). Table I also shows that all tumors induced in the glandular stomach were adenocarcinomas. Submucosal cancers were slightly less numerous in Group 3 than in Group 1. Treatment of SHR with muscimol (Group 4) slightly increased the incidence of submucosal tumors compared with that in Group 3. However, these differences were not statistically significant. Treatment of SHR with muscimol also had little or no influence on the size of the gastric cancers. There was no significant difference in the histological types of gastric cancer in the 4 groups. All cancers were found in t h e antral mucosa, and no metastases were seen ir, any rats. Tissue catecholarniize and labeling index Table I1 summarizes data on the NE concentrations in the gastric wall and the labeling indices of the gastric mucosa in each group at weeks 30 and 52. At both times, the tissue NE
TABLE I - INCIDENCE AND HISTOLOGICAL TYPES OF GASTRIC CANCER IN MNNG-TREATED RATS
(ir,iup
Strain and lruatment’
numhcr
~
1 2 3 4
_
_
WKY WKY + muscimol SHR SHR + muscimol
B(’dy weight (g)
at week 26
353? 352 5 343 5 345 4
Effective number of rats
( “h )
52
3 4 1 0 k 10 3 388 k 10 4 354 k 8’ 5 377 2 8
Number of mts with Numher of gastric gastric cancer cancers
14 15 20 20
l(7) 0 (0) 10(50)? 3 (15)4
1 0 11
Drprh of involvement (4.) ~ ~ ~ ~ Submucosal ~ l Well Muscle layer l differentiated differentiated layer or deeper Histology (4.)
l(100) OW) 9(91) 4 (80)
O(0) 0 0) 1[9)
1 (LOO) 9 (82) 5 (100)
Size of gastric cancers (mm)
O(0) -
2.0
2(18)
5.0 2 1.5 4.9 & 1.7
-
l(20) 0 (0) 5 ‘Treatment: WKY strain and SHR strain: drinking water containing MNNG for 25 weeks and then i.p. injections of 0.9% NaCI solution every other day; WKY + muscimol, and SHR + muscimol, drinking water containing MNNG for 25 weeks and then i.p. injections of 0.5 mg/kg body weight of muscimol every other day.-2JSignificantly different from the value for Group 1: ’p < 0.02, j p < 0.01.-4SSignificantly different from the value for Group 3 a t p < 0.05.
926
TATSUTA ET AL.
TABLE I1 - NOREPINEPHRINE CONCENTRATION I N THE STOMACH WALL AND LABELING INDEX O F GASTRIC MUCOSA Experi-
30
52
Group
Strain and
number
treatment'
1 2 3 4 1
2 3 4
WKY WKY + Muscimol SHR SHR + Muscimol WKY WKY + Muscimol SHR SHR + Muscimol
Norepinephrine (ngig tissue)
Labeline index ( % I
Fundic portion
Antral portion
317 2 26 342 t 14 649 t 632 611 c 86 295 2 24 303 f 14 650 2 611 588 f 47
314 ? 10 284 ? 24 498 2 23' 355 c 114 319
&
9
310 2 8 506 t 24' 355 2 114
Fundic rnucosa
Antral mucosa
10.7 ? 0.6 10.8 f 0.5 16.6 c 0A3 16.4 2 0.9 10.4 f 0.5 10.8 f 0.4 23.0 ? 1.33 20.4 2 0.8
14.3 c 0.9 13.7 f 1.0 23.2 2 0.8' 13.2 5 0.74 15.5 c 0.9 14.9 c 0.8 24.0 c 0.8? 14.8 c l.04
'For explanation of treatments, see Table I.-2,3Significantlydifferent from the value for Group 1: ?p < 0.01, 3p < 0.001.-4Significantly different from the value for Group 3 a t p < 0.001. concentrations in the fundic and antral portions of the stomach were significantly higher in Group 3 (SHR) than in Group 1 (WKY). Treatment of SHR with muscimol significantly decreased the NE concentration of the antral portion, but not the fundic portion, of the gastric wall in Group 3 at weeks 30 and 52. Treatment of WKY rats with muscimol had little or no influence on the N E concentration in the gastric wall. Epinephrine was not detected in any samples from the gastric wall at either time. At weeks 30 and 52, the labeling indices of the fundic and antral mucosa were significantly higher in Group 3 (SHR) than in Group 1 (WKY). Treatment of SHR with muscimol significantly decreased the labeling index of the antral mucosa, but not of the fundic mucosa. Treatment of WKY rats with muscimol had little or no influence on the labeling index of the gastric mucosa. DISCUSSION
The present study showed that prolonged administration of a GABAA receptor agonist muscimol attenuated the enhancement of MNNG-induced gastric carcinogenesis in SHR, resulting in a significant reduction in the incidence of gastric cancers at week 52. The mechanism(s) of this effect of muscimol is not yet known, but 3 possibilities may be considered. One explanation involves the control of anterior pituitary hormones. GABA is found in high concentrations in the hypothalamus (Miguez and Aldegunde, 1990). Inhibition of GABA degradation and blockage of GABA transmission can lead to alterations in pituitary hormone secretion (McCann et al., 1984). These facts suggest that GABA is important in physiological control of anterior pituitary hormone secretion. Miguez and Aldegunde (1990) found that GABA inhibits ACTH secretion mediated by activation of a , and a2-adrenergic receptors. There are many contradictory reports on the effect of ACTH on growth of the gastrointestinal tract. Rasanen (1963) studied the effects of ACTH on the mitotic index of rat gastric and duodenal mucosa, finding that multiple low doses of ACTH (2 IU for 4 days) caused a slight increase in the number of mitoses in the stomach but not in the duodenum. A second possibility involves an effect on the parasympathetic nervous system. Peripheral GABA receptors have been found in a variety of tissues. In the gut, GABA neurons within the myenteric plexus have been demonstrated (Jessen et al., 1983). There are 2 pharmacologically distinct types of GABA receptor that modulate the release of neurotransmitter. The activation of GABAA receptors leads to increased release of acetylcholine and contraction of longitudinal muscle strips of guinea-pig ileum (Kleinrock and Kilbinger, 1983). Rattan and Mangat (1989) reported that injection of GABA and muscimol
into the third ventricle inhibited acetylcholinesterase in the hypothalamic nuclei, and reduced the serum acetylcholinesterase activity. Acetylcholine is a neurotransmitter and its capacity to influence the development of gastrointestinal cancer has been documented. Gurkalo and Volfson (1982) found that nicotine enhances and atropine stimulates the effects of MNNG on development of gastric cancer. We showed previously that administration of the acetylcholinesterase inhibitor neostigmine every other day after MNNG treatment resulted in a significant decrease in the incidence of gastric cancer induced by MNNG, and that prolonged blockage of cholinoceptor activity by atropine resulted in a significant increase in the number of gastric cancers (Tatsuta et al., 1989a). A third possibility involves the reduction in the activity of the sympathetic nervous system by muscimol. Studies have confirmed that GABA and muscimol lower the blood pressure in SHR, and that central GABAergic dysfunction may contribute to development of spontaneous hypertension (Sasaki et al., 1990). The mechanism by which muscimol lowers the blood pressure is unknown, but may involve a decrease in the activity of the sympathetic nervous system. Sasaki et al. (1990) also found that in S H R the central GABAergic system is impaired and concluded that chronic treatment with muscimol can attenuate development of spontaneous hypertension by reducing hypothalamic over-activity. In the present work, we found that administration of muscimol resulted in a significant decrease in the norepinephrine concentration in the antral portion of the gastric wall. As the norepinephrine concentration is considered to be a marker of activity of the sympathetic nervous system, this finding suggests that administration of muscimol reduced activity of the sympathetic nervous system in the stomach of SHR rats. Evidence has been accumulating to support the concept of neural control of cell proliferation in various cells, including those in rat jejunal crypts and rat colonic crypts (Kennedy et al., 1983): alpha-adrenergic agents promote cell division (Tutton and Helme, 1974). In agreement with this concept, we found, in the present work, that prolonged administration of muscimol to S H R significantly decreased both the norepinephrine concentration in the antral portion of the gastric wall and the labeling index of the antral mucosa. There is a strong positive correlation between the mortality from gastric cancer and diseases related to hypertension, such as cerebrovascular disease, in different countries (Sonnenberg, 1988). Both diseases are frequent in Japan and Chile, and rare in America and Canada (Whelton and Goldblatt, 1982). Japan, with a high mortality from gastric cancer, showed a conspicuously high mortality from cerebrovascular disease (Sato et al., 1959). In most countries, the mortalities from gastric cancer and cerebrovascular disease have declined in a parallel manner during the past 30 years (Tuomilehto ef al.,
GASTRIC CANCER INHIBITION BY MUSCIMOL IN SHR
1984). Sonnenberg (1988) analyzed data from the German social security system to determine whether ischemic heart disease and cerebrovascular disease occurred in patients with gastric cancer more frequently than could be explained by chance alone. He found that gastric cancer occurred more frequently in patients who had concomitant ischemic heart disease or cerebrovascular disease, and concluded that gastric cancer and diseases related to hypertension share a common etiologic factor. The present results show that prolonged administration of muscimol attenuated the enhancement of gastric carcinogene-
927
sis by MNNG in SHR and suggested that this effect of muscimol may be closely related to its effect in reducing the activity of the sympathetic nervous system and subsequently decreasing proliferation of the antral epithelial cells.
ACKNOWLEDGEMENTS
This work was supported in part by a Grant-in-Aid from the Ministry of Health and Welfare for a Comprehensive 10-Year Strategy for Cancer Control, Japan.
REFERENCES EASTWOOD,G.L. and QUIMBY, G., Effect of chronic cimetidine ingestion on fundic and antral epithelial proliferation in the rat. Dig. Dis. Sci., 28, 61-64 (1983). GURKALO,V.K. and VOLFSON,N.J., Nicotine influence upon the development of experimental stomach tumors. Arch. Geschwulstforsch., 4,259-265 (1982). JESSEN,K.R., HILLS,J.M., DENNISON, M.E. and MIRSKY,R., Gammaaminobutyrate as an autonomic neurotransmitter: release and uptake of ['Hlgamma-aminobutyrate in guinea-pig large intestine and cultured enteric neurons using physiological methods and electron microscopic autoradiography. Neuroscience, 10, 1427-1442 (1983). KENNEDY, M.F.G., TUTTON,P.J.M. and BARKLA,D.H., Adrenergic factors involved in the control of crypt cell proliferation in jejunum and ascending colon of mouse. Clin. exp. Pharmacol. Physiol., 10, 577-586 (1983).
KLHNROCK, A. and KILBINGER, H., Gamma-aminobutyric acid and cholinergic transmission in the guinea-pig ileum. Naunyn-Schmideberg's Arch. exp. Pharmakol., 322,216-220 (1983). MC'CANN,S.M., VIJAYAN. E., NEGRO-VILAR, A,, MIZUNUMA, H. and MANGA-r, H.. Gamma aminobutyric acid (GABA), a modulator of anterior pituitary hormone secretion by hy othalamic and pituitary action. PJ).choneuroendocrinology,9,97-106 6984). MIGUEZ,I. and ALDEGUNDE, M.A., Effect of gamma-aminobutyric acid on corticosterone secretion: involvement of the noradrenergic system. Life Sci., 46,875-880 (1990). MILLER, R.G., JR., Simultaneous statistical inference. McGraw-Hill, New York (1966). MORSTYN, G.H., Hsu, S.M., KINSELLA, T., GRATZNER,H., Russo, A. and MITCHELL, J.P., Bromodeoxyuridine in tumors and chromosomes detected with a monoclonal antibody. J. din. Invest., 72, 1844-1850 (1983).
RASANEN, T., Fluctuations in the mitotic frequency of the glandular stomach and intestine of the rat under the influence of ACTH, glucocorticoids, stress and heparin. Acta physiol. scand., 58, 211-220 (1963).
RATTAN,A.K. and MANGAT,H.K., Effect of GABA, muscimol and
picrotoxin given in third ventricle on serum cholinesterases and monoamine oxidase and on plasma succinic dehydrogenase in rat. Ind. J. Physiol. Pharmacol., 33,203-206 (1989). SASAKI,S., NAKATA,T., KAWASAKI, S., HAYASHI,J., OCURO,M., TAKEDA,K. and NAKAGAWA, M., Chronic central GABAergic stimulation attenuates hypothalamic hyperactivity and development of spontaneous hypertension in rats. J. cardiovasc. Pharmacol., 15, 706-713 (1990).
SATO,T., FUKUYAMA, T. and SUZUKI,T., Studies of the causation of gastric cancer. 2. The relation between gastric cancer mortality rate and salted food intake in several places in Japan. Bull. Inst. publ. Hlth, 8,187-198 (19.59).
SONNENBERG, A,, Concordant occurrence of gastric and hypertensive diseases. Gastroenterology,95,4248 (1988). TATSUTA,M.. IISHI, H. and BABA,M.. Inhibition by neostigmine and isoproterenol and promotion by atropine of experimental carcinogenesis in rat stomach by N-methyl-N'-nitro-N-nitrosoguanidine. Int. J. Cancer, 44,188-189 (1989a). TATSUTA, M., IISHI,H.. BABA,M. and TANIGUCHI, H., Enhancement of experimental gastric carcinogenesis induced in spontaneously hypertensive rats by N-methyl-N'-nitro-N-nitrosoguanidine. Cancer Rex, 49, 794-798 (1989b).
I., GEBOERS,J., JOOSSENS.J.V., SALONEN,J.T. and TANSKANEN, T.. Trends in stomach cancer and stroke in Finland. Comparison to northwest Europe and USA. Stroke, 15, 823-828
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(1984).
TUTTON,P.J.M. and HELME,R.D.. The influence of adrenoceptor activity on crypt cell proliferation in the rat jejunum. Cell Tissue Kinet., 7, 125-136 (1974). UNGER,T., BECKER,H., DIETZ,R., GANTEN, D., LANG,R.E., RETTINC, R., SCHOMIG,A. and SCHWAB,N.A., Antihypertensive effect of the GABA receptor agonist muscimol in spontaneously hypertensive rats. Role of the sympathoadrenal axis. Circul. Res., 54,30-37 (1984). WHELTON,P.K. and GOLDBLATT. P., An investigation of the relationship between stomach cancer and cerebrovascular disease. Amer. J. Epidemiol., 115,418-427 (1982).
%b :
Publication of the International Union Against Cancer Publication de I Union Internationale Contre le Cancer
PROTECTION BY MUSCIMOL AGAINST GASTRIC CARCINOGENESIS INDUCED BY N-METHYL-" -NITRO-N-NITROSOGUANIDINE IN SPONTANEOUSLY HYPERTENSIVE RATS Masaharu TATSUTA' ', Hiroyasu I I S H I ~Miyako , BABA',Hiroyuki UEHARA', Akihiko NAKAIZUMI' and Haruo T A N I G U C H I ~ Departments of 'Gastrointestiiial Oncology and ?Pathology, The Center for Adult Disease, Obaka, 3-3, Nakumichi I-chome, Higash~nan-kir,Osaka 537, Japan. The effects of prolongedadministration of the gamma-aminobutyric acid receptor agonist muscimol on enhanced induction of gastric carcinogenesis by N-methyl-N'-nitro-N-nitrosoguanid ine (MNNG) in spontaneously hypertensive rats (SHR), and on the norepinephrine concentration in the gastric wall and the labeling index of gastric mucosa were investigated. SHR and normotensive Wistar Kyoto (WKY) rats as controls were given a solution of MNNG (25 pg/ml) for 25 weeks and then i.p. injections of 0.5 mg/kg body weight of muscimol every other day. In control WKY rats, gastric cancers were found in I (7%) of 14 rats examined a t week 52. In SHR treated with NaCl solution only, the incidence of gastric cancers was significantly increased to 50% compared with that in control WKY rats. However, treatment of SHR with muscimol significantly increased its incidence to 12% compared with the value in SHR treated with NaCl solution only. The norepinephrine concentration in the gastric wall and the labeling index of the gastric mucosa were significantly greater in SHR than in WKY rats. Prolonged administration of muscimol to SHR significantly reduced the norepinephrine concentration in the antral portion of the gastric wall or the labeling index of the antral epithelial cells. These findings indicate that long-term treatment of SHR with muscimol attenuated the enhancement of gastric carcinogenesis in SHR.
o 1992 Wilq-Liss, Inc. We have found that, at week 52, the incidence and number of gastric cancers induced by MNNG were significantly greater in SHR than in WKY rats. and that the norepinephrinc concentration of the gastric wall was also higher in SHR (Tatsuta et a/., 1989b). As the norepinephrine concentration is considered to be a marker of the sympathetic nervous system, this tinding suggested that an increase in sympathetic nervous system activity in SHR may enhance the process of gastric carcinogenesis. Unger et 01. (1984) found that injection of the potent GABA agonist muscimol into the lateral brain ventricle lowered the mean arterial blood pressure dose-dependently in SHR, and suggested that this was due to selective suppression of sympathoadrenal activity. Their results indicated that administration of muscimol might attenuate gastric carcinogenesis in SHR, and therefore, in the present work, we examined its effect on gastric canccr induced by MNNG in SHR.
MATERIAL AND METHODS
Animal5 Six-week-old male SHR and age-matched male WKY rats were obtaincd from Hoshino (Tochigi, Japan). SHR were from the colony of the Department of Pharmacology, Jichi Medical School, Tochigi, Japan. In this study, WKY rats were used as controls for SHR. SHR were originally derived from WKY rats and thus these 2 strains had the same genetic origin but not the same hypersensitivity. The rats were housed in suspended, wire-bottomcd metal cages in animal quarters at controlled temperature (21-22°C) and humidity (30-50%), with a 12-hr light, 12-hr dark cycle. and were given free access to regular chow pellets (Oriental Yeast, Tokyo, Japan).
Experimental design Rats of the SHR and WKY strains were given drinking water containing MNNG (25 pg/ml; Aldrich, Milwaukee, WI) for 25 weeks. MNNG was dissolved in deionized water at a concentration of 2 mgikg and kept in a cool, dark place and the stock solution was diluted to 25 pgiml with tap water just before use. Each rat was given 40 ml of MNNG solution (less than a single rat can consume in 48 hr) from a bottle covered with aluminum foil to prevent photolysis of MNNG, and the solution was replenished every other day. At week 26, the WKY and SHR rats were each randomly divided into 2groups, and treated as follows until the end of the experiment at week 52. One group of WKY rats (Group 1: 25 rats) and one group of SHR (Group 3: 30 rats) were given i.p. injections of 2 ml/kg body weight of 0.9% NaCl solution every other day. The other groups of WKY rats (Group 2; 25 rats) and SHR (Group 4; 30 rats) were given i.p. injections of 0.5 mgikg body weight of muscimol every other day. Muscimol (Sigma, St. Louis, MO) in 0.9% NaCl solution was injected in a volume of 2 mlikg body weight. Injections were given between 2 and 3 p.m.
Histological observations Ten rats each from each group were killed at week 30 for measurement of the catecholamine concentration of the gastric wall and the labcling index of the gastric mucosa. Animals that survived for more than 50 weeks were included in effective numbers because the first tumor of the glandular stomach was found in a rat in Group 3 that died at week 50. All surviving animals were killed at the end of the experiment at week 52 and autopsied. The stomach and other organs were carefully examined. The stomach was opened along the greater curvature, pinned flat on a cork mat, and fixed with Zamboni's solution for histological examination. The fixed stomach was cut into longitudinal strips, 3 mm wide. The specimens were then embedded in paraftin, and serial sections 5 pm thick were stained with hcmatoxylin and eosin. Sections were examincd without knowledge of which group they were from. Classification qf gastric cancers Histologically, adenocarcinomas were defined as lesions in which neoplastic cells had penetrated the muscularis mueosae to involve the submucosa or deeper layers. Adenocarcinomas were classified as very well-differentiated, well-differentiated, and poorly differentiated types. Meusurement of cutecholamivies in the gastric wall The epinephrine and NE concentrations in the tissues of the gastric wall were determined by high-performance liquid chromatography as reported previously (Tatsuta et al., 19896) 3Towhom correspondence and reprint requests should be sent A hbreviations: MNNG, N-methyl-N'-nitro-N-nitrosoguanidine, BrdU,
hromodeoxyuridine;NE, norepinephrine;muscimol, 5-arninomethyl-3hydroxyisoxazole. ~~
Received: May 18, 1992 and in revised form August 11, 1992
925
GASTRIC CANCER INHIBITION BY MUSCIMOL IN SHR
at weeks 30 and 52. For this purpose, 5 rats in each group were starved for 12 hr and then given 2 mlikg of 0.9% NaCl solution (Groups 1 and 3) or 0.5 mgikg of muscimol (Groups 2 and 4). Two hours later, the rats were killed by cervical dislocation. Samples of about 50 mg of the wall of the fundic and antral portions o f the stomach were removed from each rat, homogenized with 4 ml of 0.4 N perchloric acid, and centrifuged at 1,lOOg for 10 min. The supernatant was mixed with 1 ml of 0.2 M disodium ethylenediaminetetraacetate (EDTA), and the mixture was adjusted to p H 6.0 with ammonium hydroxide. Then the mixture was added to 300 mg of purified aluminum (Woelm Neutral Active Grade I), and the pH was adjusted to 8.4-8.8 with ammonium hydroxide. The mixture was stirred for 5 min and centrifuged at 10,000 g for 10 min, then the supernatant was aspirated and discarded. The precipitated aluminum was washed twice with distilled water and then shaken vigorously with 2.5 ml of 0.4 N acetic acid. The mixture was centrifuged, the clear supernatant was transferred to a small glass tube and lyophilized for 3 hr, and the residue was dissolved in 0.5 ml of 0.2 N acetic acid. A 50-)*I aliquot of this solution was injected into a liquid chromatographic column (Hitachi 301 I-C gel column, 2.6 X 250 mm). Materials were eluted with 0.1 M KHzPOI containing 0.05% HIPOj at a constant flow rate of 0.5 mlimin at 45.0 ? 0.2”C. The eflluent was mixcd with the reagent for the trihydroxyindol reaction, consisting of 0.0075% potassium ferricyanide, 0.1% ascorbic acid, and 5 N sodium hydroxide. The resulting fluorescent products were measured with a highly sensitive spectrofluorophotometer.
Measrircwient of labeling index of gastric mucosa The labeling index of the gastric mucosa was examined at weeks 30 and 52. The labeling index was measured with an immunohistochemical analysis kit (Becton-Dickinson, Mountain View, CA) for assaying BUdR incorporation (Morstyn et ul.. 1983). For this purpose, 5 rats in each group were starved for 12 hr and then treated i.p. with either 2 mlikg of 0.9% NaCl solution (Groups 1 and 3) or 0.5 mgikg of muscimol (Groups 2 and 4). One hour later, BUdR (20 mgikg) was injected i.p., and after another hour the animals were killed with ether. The stomach was removed and fixed in 70% ethanol for 4 hr, and then cmbcdded in paraffin. Thin sections 3 km thick were immersed in 2 N HCI solution for 30 min and then in 0.1 M Na2B407.Slides were also immersed on 0.3% H20,in methanol for 30 min to block endogenous peroxidase activity, and then treated with 10%’ horse serum. Specimens were incubated with anti-BUdR monoclonal antibody (diluted 1:20) for 2 hr, washed, and stained first with biotin-conjugated horse antimouse antibody (Vector, Burlingame, CA: diluted 1:200) for 30 niin. and then with avidin-biotin-peroxidase complex (Vector) for 30 min. The reaction product was detected with 3,3’-dianiinobcnzidine tetrahydrochloride. Cells that contained BUdR were identified by the presence of a dark pigment over their nucleus. To determine the labeling index of
the gastric mucosa, the numbers of BUdR-labeled and -unlabeled cells in the zone of proliferating cells were counted (Eastwood and Quimby, 1983) without knowledge of which treatment group the samples were from. The zone of proliferating cells in the fundic mucosa was defined as a 250-pm rectangular area between the highest and lowest cells in a well-oriented section. Ten such rectangular areas of each rat were examined. In the antral mucosa, all cells below the highest labeled cell in each pit-gland column were regarded as being within the zone of proliferating cells, and 100 welloriented pit-gland columns in each rat were examined. On the basis of these measurements, we calculated the labeling index as the number of BUdR-labeled cellsitotal number of cells within the proliferating zone.
Statistical analysis Results were analyzed by the Chi-square test or Fisher’s exact probability test, or by one-way analysis of variance with Dunn’s multiple comparison (Miller, 1966). Data are shown as means 5 SE. “Significant” indicates a calculated p-value of less than 0.05. RESULTS
Incidence and histological ppes of gastric cancer One rat in Group 1 died before week 35. No tumors were found in this animal, which was excluded from the effective number. At week 52, the body weights of the SHR group without muscimol treatment were significantly lower than those of the other groups. The incidence and histological type of gastric cancers in MNNG-treated rats are summarized in Table I . In control Group I (WKY), gastric cancers were found in only one (7%) of 14 rats examined. In Group 3 (SHR), the incidence of gastric cancers was significantly higher than in Group 1. Treatment of SHR with muscimol resulted in a significant decrease in the incidence of gastric cancers (Group 4 vs. Group 3). Table I also shows that all tumors induced in the glandular stomach were adenocarcinomas. Submucosal cancers were slightly less numerous in Group 3 than in Group 1. Treatment of SHR with muscimol (Group 4) slightly increased the incidence of submucosal tumors compared with that in Group 3. However, these differences were not statistically significant. Treatment of SHR with muscimol also had little or no influence on the size of the gastric cancers. There was no significant difference in the histological types of gastric cancer in the 4 groups. All cancers were found in t h e antral mucosa, and no metastases were seen ir, any rats. Tissue catecholarniize and labeling index Table I1 summarizes data on the NE concentrations in the gastric wall and the labeling indices of the gastric mucosa in each group at weeks 30 and 52. At both times, the tissue NE
TABLE I - INCIDENCE AND HISTOLOGICAL TYPES OF GASTRIC CANCER IN MNNG-TREATED RATS
(ir,iup
Strain and lruatment’
numhcr
~
1 2 3 4
_
_
WKY WKY + muscimol SHR SHR + muscimol
B(’dy weight (g)
at week 26
353? 352 5 343 5 345 4
Effective number of rats
( “h )
52
3 4 1 0 k 10 3 388 k 10 4 354 k 8’ 5 377 2 8
Number of mts with Numher of gastric gastric cancer cancers
14 15 20 20
l(7) 0 (0) 10(50)? 3 (15)4
1 0 11
Drprh of involvement (4.) ~ ~ ~ ~ Submucosal ~ l Well Muscle layer l differentiated differentiated layer or deeper Histology (4.)
l(100) OW) 9(91) 4 (80)
O(0) 0 0) 1[9)
1 (LOO) 9 (82) 5 (100)
Size of gastric cancers (mm)
O(0) -
2.0
2(18)
5.0 2 1.5 4.9 & 1.7
-
l(20) 0 (0) 5 ‘Treatment: WKY strain and SHR strain: drinking water containing MNNG for 25 weeks and then i.p. injections of 0.9% NaCI solution every other day; WKY + muscimol, and SHR + muscimol, drinking water containing MNNG for 25 weeks and then i.p. injections of 0.5 mg/kg body weight of muscimol every other day.-2JSignificantly different from the value for Group 1: ’p < 0.02, j p < 0.01.-4SSignificantly different from the value for Group 3 a t p < 0.05.
926
TATSUTA ET AL.
TABLE I1 - NOREPINEPHRINE CONCENTRATION I N THE STOMACH WALL AND LABELING INDEX O F GASTRIC MUCOSA Experi-
30
52
Group
Strain and
number
treatment'
1 2 3 4 1
2 3 4
WKY WKY + Muscimol SHR SHR + Muscimol WKY WKY + Muscimol SHR SHR + Muscimol
Norepinephrine (ngig tissue)
Labeline index ( % I
Fundic portion
Antral portion
317 2 26 342 t 14 649 t 632 611 c 86 295 2 24 303 f 14 650 2 611 588 f 47
314 ? 10 284 ? 24 498 2 23' 355 c 114 319
&
9
310 2 8 506 t 24' 355 2 114
Fundic rnucosa
Antral mucosa
10.7 ? 0.6 10.8 f 0.5 16.6 c 0A3 16.4 2 0.9 10.4 f 0.5 10.8 f 0.4 23.0 ? 1.33 20.4 2 0.8
14.3 c 0.9 13.7 f 1.0 23.2 2 0.8' 13.2 5 0.74 15.5 c 0.9 14.9 c 0.8 24.0 c 0.8? 14.8 c l.04
'For explanation of treatments, see Table I.-2,3Significantlydifferent from the value for Group 1: ?p < 0.01, 3p < 0.001.-4Significantly different from the value for Group 3 a t p < 0.001. concentrations in the fundic and antral portions of the stomach were significantly higher in Group 3 (SHR) than in Group 1 (WKY). Treatment of SHR with muscimol significantly decreased the NE concentration of the antral portion, but not the fundic portion, of the gastric wall in Group 3 at weeks 30 and 52. Treatment of WKY rats with muscimol had little or no influence on the N E concentration in the gastric wall. Epinephrine was not detected in any samples from the gastric wall at either time. At weeks 30 and 52, the labeling indices of the fundic and antral mucosa were significantly higher in Group 3 (SHR) than in Group 1 (WKY). Treatment of SHR with muscimol significantly decreased the labeling index of the antral mucosa, but not of the fundic mucosa. Treatment of WKY rats with muscimol had little or no influence on the labeling index of the gastric mucosa. DISCUSSION
The present study showed that prolonged administration of a GABAA receptor agonist muscimol attenuated the enhancement of MNNG-induced gastric carcinogenesis in SHR, resulting in a significant reduction in the incidence of gastric cancers at week 52. The mechanism(s) of this effect of muscimol is not yet known, but 3 possibilities may be considered. One explanation involves the control of anterior pituitary hormones. GABA is found in high concentrations in the hypothalamus (Miguez and Aldegunde, 1990). Inhibition of GABA degradation and blockage of GABA transmission can lead to alterations in pituitary hormone secretion (McCann et al., 1984). These facts suggest that GABA is important in physiological control of anterior pituitary hormone secretion. Miguez and Aldegunde (1990) found that GABA inhibits ACTH secretion mediated by activation of a , and a2-adrenergic receptors. There are many contradictory reports on the effect of ACTH on growth of the gastrointestinal tract. Rasanen (1963) studied the effects of ACTH on the mitotic index of rat gastric and duodenal mucosa, finding that multiple low doses of ACTH (2 IU for 4 days) caused a slight increase in the number of mitoses in the stomach but not in the duodenum. A second possibility involves an effect on the parasympathetic nervous system. Peripheral GABA receptors have been found in a variety of tissues. In the gut, GABA neurons within the myenteric plexus have been demonstrated (Jessen et al., 1983). There are 2 pharmacologically distinct types of GABA receptor that modulate the release of neurotransmitter. The activation of GABAA receptors leads to increased release of acetylcholine and contraction of longitudinal muscle strips of guinea-pig ileum (Kleinrock and Kilbinger, 1983). Rattan and Mangat (1989) reported that injection of GABA and muscimol
into the third ventricle inhibited acetylcholinesterase in the hypothalamic nuclei, and reduced the serum acetylcholinesterase activity. Acetylcholine is a neurotransmitter and its capacity to influence the development of gastrointestinal cancer has been documented. Gurkalo and Volfson (1982) found that nicotine enhances and atropine stimulates the effects of MNNG on development of gastric cancer. We showed previously that administration of the acetylcholinesterase inhibitor neostigmine every other day after MNNG treatment resulted in a significant decrease in the incidence of gastric cancer induced by MNNG, and that prolonged blockage of cholinoceptor activity by atropine resulted in a significant increase in the number of gastric cancers (Tatsuta et al., 1989a). A third possibility involves the reduction in the activity of the sympathetic nervous system by muscimol. Studies have confirmed that GABA and muscimol lower the blood pressure in SHR, and that central GABAergic dysfunction may contribute to development of spontaneous hypertension (Sasaki et al., 1990). The mechanism by which muscimol lowers the blood pressure is unknown, but may involve a decrease in the activity of the sympathetic nervous system. Sasaki et al. (1990) also found that in S H R the central GABAergic system is impaired and concluded that chronic treatment with muscimol can attenuate development of spontaneous hypertension by reducing hypothalamic over-activity. In the present work, we found that administration of muscimol resulted in a significant decrease in the norepinephrine concentration in the antral portion of the gastric wall. As the norepinephrine concentration is considered to be a marker of activity of the sympathetic nervous system, this finding suggests that administration of muscimol reduced activity of the sympathetic nervous system in the stomach of SHR rats. Evidence has been accumulating to support the concept of neural control of cell proliferation in various cells, including those in rat jejunal crypts and rat colonic crypts (Kennedy et al., 1983): alpha-adrenergic agents promote cell division (Tutton and Helme, 1974). In agreement with this concept, we found, in the present work, that prolonged administration of muscimol to S H R significantly decreased both the norepinephrine concentration in the antral portion of the gastric wall and the labeling index of the antral mucosa. There is a strong positive correlation between the mortality from gastric cancer and diseases related to hypertension, such as cerebrovascular disease, in different countries (Sonnenberg, 1988). Both diseases are frequent in Japan and Chile, and rare in America and Canada (Whelton and Goldblatt, 1982). Japan, with a high mortality from gastric cancer, showed a conspicuously high mortality from cerebrovascular disease (Sato et al., 1959). In most countries, the mortalities from gastric cancer and cerebrovascular disease have declined in a parallel manner during the past 30 years (Tuomilehto ef al.,
GASTRIC CANCER INHIBITION BY MUSCIMOL IN SHR
1984). Sonnenberg (1988) analyzed data from the German social security system to determine whether ischemic heart disease and cerebrovascular disease occurred in patients with gastric cancer more frequently than could be explained by chance alone. He found that gastric cancer occurred more frequently in patients who had concomitant ischemic heart disease or cerebrovascular disease, and concluded that gastric cancer and diseases related to hypertension share a common etiologic factor. The present results show that prolonged administration of muscimol attenuated the enhancement of gastric carcinogene-
927
sis by MNNG in SHR and suggested that this effect of muscimol may be closely related to its effect in reducing the activity of the sympathetic nervous system and subsequently decreasing proliferation of the antral epithelial cells.
ACKNOWLEDGEMENTS
This work was supported in part by a Grant-in-Aid from the Ministry of Health and Welfare for a Comprehensive 10-Year Strategy for Cancer Control, Japan.
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