Inhibitory effect of magnesium hydroxide on

Carcinogenesls vol.10 no.3 pp.613-616, 1989

SHORT COMMUNICATION

Inhibitory effect of magnesium hydroxide on methylazoxymethanol acetate-induced large bowel carcinogenesis in male F344 rats

Takuji Tanaka1, Tokuro Sbinoda, Naoki Yoshimi, Kenji Niwa, Hitoshi Iwata and Hideki Mori 1st Department of Pathology, Gifu University School of Medicine, 40 Tsukasa-machi, Gifu City 500, Japan 'To whom reprint requests should be sent

It is well-known that nutritional factors play a significant role in the development of neoplasms. Chemoprevention is a concept that certain noncarcinogenic synthetic chemicals or naturally occurring products in some food inhibit the process of carcinogenesis. Until now, certain chemopreventive agents proved effective on chemical carcinogenesis in different organs (1). Trace metals and metals are known to have some inhibitory effects on tumor induction and development (2). For example, selenium (3,4) or a newly synthesized organo-selenium compound, />-methoxybenzeneselenol (5,6) is reported to have anticarcinogenic properties in several target organs including colon. Although a number of publications have noted that trace elements and metals modify enzymes and other subcellular components, the relevance of these observations to the carcinogenic process has been evaluated on only a few occasions. Durlach et al. have reviewed the role of magnesium on the induction of neoplasia, postulating a possibility that magnesium may display an antitumorigenic effect (7). In experimental studies, an increased incidence of lymphoma or thymoma was observed in rats kept on a magnesium deficient diet (8,9). Bazikyan and Akimov (10) found that magnesium chloride in the drinking water (15 mg/kg) suppressed skin tumorigenesis by 1,2,5,6-dibenzanthracene. Some clinical studies report that magnesium administration has an effect in the treatment of breast cancer (2). Magnesium as well as calcium is also known to be a fundamental regulator of the cell cycle (11). Although the mechanism is not clear, the major essential metals, magnesium and calcium, have inhibited tumor induction by heavy metals (12-14). However, these experimental data record local inhibition (12,14) and few

•Abbreviations: MAM, methylazoxymethanol © IRL Press

Table I. Percentage composition of basal diet, CE-21 Ingredient Crude Crude Crude Ash Crude Water

carbohydrate1' protein0 fat"1 cellulosee

Percentage 49.6 25.3 4.8 7.4 3.7 9.2

•345.2 Cal; Ca 1.22%, P 1.18%; the content of vitamins, A, D3, E, B,, and B 6 are 10 and 2 IU, and 51, 16, 14 and 16 jig/g diet. "The main sources of crude carbohydrate are corn, wheat and rice-bran. T h e main sources of crude protein are fish meal and soybean cake. d The main sources of crude fat are corn oil and lard. °No special material was added as a source of crude cellulose.

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The effect of dietary magnesium hydroxide on colon carcinogenesis induced by methylazoxymethanol (MAM) acetate was examined in male F344 rats. MAM acetate was administered by i.p. injection to rats at 25 mg/kg body wt once per week for 3 weeks. Starting 2 weeks after the final MAM acetate exposure, the diet containing 500 or 1000 p.p.m. magnesium hydroxide was fed for 227 days. In the groups receiving magnesium hydroxide and MAM acetate, the incidence of colon neoplasms was decreased when compared with that in the group given MAM acetate alone. The inhibitory effect of dietary magnesium hydroxide on MAM acetate-induced colon carcinogenesis was greater at the lower dose than that at the higher dose of magnesium hydroxide hi the diet. Neoplasms in other organs were rare and were not affected by the dietary magnesium hydroxide.

systemic effects have been demonstrated (13,14). Some data relate magnesium concentrations in water, food and air to cancer mortality (15-19) and epidemiological investigations have also suggested that magnesium is related to a low cancer mortality of people living at high altitudes (20-22). Furthermore, several recent reviews have dealt with an anticarcinogenic effect of magnesium (1,2,7,23,24). In the present study, the effect of magnesium on the development of colon neoplasms was examined in a rat model with use of methylazoxymethanol (MAM*) acetate and magnesium hydroxide. A total of 155 male F344 rats, 5-weeks-old, from Shizuoka Laboratory Animal Center (Shizuoka, Japan) were quarantined for 7 days and randomized by weight into six groups. All rats were kept in a temperature (23 ± 2°C), humidity (50 ± 10%) and light (12 h light/dark cycle) controlled animal room and housed three or four to a wire cage. Drinking water was provided in a bottle. At 6 weeks of age, rats in groups 1, 2 and 3 were given i.p. injections of MAM acetate (25 mg/kg body wt, purchased by Sigma Chemical Co., St Louis, MO) once a week for 3 weeks. Two weeks after the final MAM acetate treatment, rats in groups 2 and 3 were transferred to the diet containing 500 p.p.m. or 1000 p.p.m. magnesium hydroxide (purity >98%, from Tateho Chem. Indust. Co., Ltd, Akoh, Japan), and kept on the diet until the end of the experiment. Animals in groups 4 and 5 received an equal volume of normal saline and given the diets with magnesium hydroxide at concentrations 500 p.p.m. and 1000 p.p.m., respectively, as for groups 2 and 3. Rats in groups 1 and 6 were continued on the basal diet, CE-2 (Table I, purchased by CLEA Japan Inc., Tokyo, Japan) throughout the experiment. All rats were provided with water and the diets ad libitum. Animals were weighed weekly until they reached 14 weeks of age, and then every 4 weeks. The experiment was terminated after 255 days after the first MAM acetate dose. Complete necropsies were performed on animals that were dying or moribund, and on all remaining animals at the termination of the experiment. All organs, especially the intestine, were carefully inspected grossly and all abnormal lesions were examined

T.Tanaka et al.

Table n . Mean survival time, body weight, liver weight and relative liver weight of rats in each group Group no.

Treatment

No. of effective rats'

Mean survival (days) of effective rats

Body weight (g)

Liver weight (g)

Relative liver weight

1 2

MAM acetate MAM acetate/ 500 p.p.m. Mg(OH)2 MAM acetate/ 1000 p.p.m. Mg(OH>2 500 p.p.m. Mg(OH)2 1000 p.p.m. Mg(OH)2 No treatment

32 30

250 249

343 ± 45 b 307 ± 52C

10.8 ± 3.5 10.9 ± 4.0

3.27 ± 0.50 3.25 ± 0.48

30

243

295 ± 561

9.6 ± 2.7

3.16 ± 0.68

20 19 20

244 250 253

337 ± 62 327 ± 27 344 ± 58

10.9 ± 2.6 10.1 ± 3.6 11.4 ± 3.8

3.24 ± 0.53 3.25 ± 0.63 3.65 ± 0.46

Large intestine

Kidney

3 4 5 6

"Rats that survived for more than 126 days. b Mean ean ± SD Significantly different from group 1 by Student's f-test ("7" < 0.01 and d P < 0.001). Table ffl. Incidence of neoplasms in the intestine and kidney of rats in each group Group no.

1 2

4 5 6

MAM acetate MAM acetate/ 500 p.p.m. Mg(OH)j MAM acetate/ 1000 p.p.m. 500 p.p.m. Mg(OH>2 1000 p.p.m. Mg(OH)2 No treatment

No. of rats with neoplasms (total neoplasms)

Total intestine Total

AD

32 30

18(29) 6X6)C

18(26) 5(5) d

30

12(14)

11(13)

No. of effective rats*

No. of rats with tumors (no. of tumors) at: Small intestine b

ADC

Total

AD

ADC

Total

AD

ADC

MCT

8(8) 0=

15(18) 5(5) f

2(2) 2(2)

0 0

2(2) 2(2)

17(24) 3(3)8

8(8) 0*

14(16) 3(3)'

3(3) 1(1)

3(3)

9(10)

2(2)

0

2(2)

9(11)

3(3)

7(8)

KD

20

0

0

0

0

0

0

0

0

0

0

0

19 20

0 0

0 0

0 0

0 0

0 0

0 0

0 0

0 0

0 0

0 0

0 0

"Rats that survived for more than 126 days. b AD, adenoma; ADC, adenocarcinoma; MCT, mesenchymal tumor. c "'Significantly different from group 1 by Chi-square test or Fisher's exact probability test; CP < 0.005, d/> < 0.001, CP < 0.003, 'P < 0.008, gP < 0.0002, hP < 0.003 and 'P < 0.003.

histologically. All tissues were processed for histologic examination by conventional methods and stained with hematoxylin and eosin. Intestinal tumors were diagnosed according to the criteria described by Ward (25). Renal tumors were diagnosed according to the criteria described by Hard and Butler (26). Statistical analysis of tumor incidence and multiplicity was done by Chi-square test, Fisher's exact probability test and Student's Mest. One rat from group 2, which was moribund and necropsied on the 126th day, had a colon tumor. Therefore, the rats alive on that day were counted as effective animals. The body weight of rats receiving MAM acetate with magnesium hydroxide (groups 2 and 3) was significantly lower than that of rats given MAM acetate alone (group 1) (Table II). However, there was no acute toxic effect, low survival or poor condition caused by dietary magnesium hydroxide. Macroscopically, most tumors were present in the large intestine. They were sessile or pedunculated polyps. Intestinal tumors were adenomas or adenocarcinomas with a higher incidence of adenocarcinoma. The dietary administration of magnesium hydroxide at a level of 500 p.p.m. resulted in a significant inhibition of MAM acetate-induced colon carcinogenesis as revealed by a low incidence of both adenomas and adenocarcinomas of the colon (Table HI). The incidence of colon 614

tumor in rats treated with MAM acetate and 1000 p.p.m. of magnesium hydroxide in the diet was lower than that in rats given MAM acetate alone, but the difference was not significant between the two groups. Small intestine and kidney tumor incidences did not differ significantly among the experimental groups. Renal neoplasms developed in rats of groups 1—3 were all mesenchymal tumors. In rats of groups 4—6, no neoplasms were present. The results of the present study indicate that the induction of colon neoplasms by MAM acetate was inhibited by magnesium hydroxide when it was fed after the carcinogen administration. The inhibitory effect of dietary magnesium hydroxide was greater at the lower dose (500 p.p.m.) than that at higher dose (1000 p.p.m.). We are not aware of any previous study of a potential colon tumor inhibitory effect by magnesium hydroxide. Magnesium, one of the major essential metals, is capable of preventing the induction of local tumors at the s.c. injection site of heavy metals in rats (12,14,27) and the pulmonary tumorigenesis in mice by lead or nickel (13). In these experimental models, the application of magnesium (as a magnesium salt) was mainly local. Concerning the mechanisms underlying inhibitory effects of magnesium on these local carcinogenesis by metals, several hypotheses have been advanced (12,28,29), i.e. (1) magnesium acts as an antagonist against the


3

Treatment

Effect of magnesium on development of colon neoplasms

Acknowledgements We are grateful to Dr John H.Weisburger of the Naytor Dana Institute for Disease Prevention, American Health Foundation, for helpful discussion and review of the manuscript.

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metal carcinogenicity; (2) magnesium inhibits the binding of carcinogenic metals to the target tissue. In the present study, effect of magnesium on chemical-induced colon carcinogenesis was examined by a systemic application of magnesium hydroxide. Similar studies have not yet been done, except two studies in which increased neoplasms were obtained in rodents maintained on magnesium-deficient diets and some protection was seen in rodents given excess levels of magnesium (8—10). For the mechanistic background of the inhibitory effect of magnesium hydroxide on MAM acetate-induced intestinal carcinogenesis demonstrated here, effect of magnesium hydroxide might be related to the colonic pH and transit time of fecal passage. High colonic pH is reported to promote colorectal cancer (30) and dietary fiber protects against colorectal cancer by speeding colonic transit and increasing stool weight (31). Magnesium hydroxide has a weak activity as a cathartic and is capable of altering the pH of intestinal lumen. Thus, changed pH and transit speed of stool induced by magnesium hydroxide may be one of the causes for the inhibitory effect of the agent. Meanwhile, magnesium is known to have some important biological effects (32). Magnesium activates many enzymes involved in the cell cycle (throughout the cell cycle) and oxidative phosphorylation (11,32). Furthermore, magnesium is also known as a stabilizer of the DNA, RNA and ATP structures (32). Magnesium ions can stabilize DNA binding to the phosphates on the surface of the molecule and allow incorporation of only the proper nucleotides during DNA synthesis (33,34). Magnesium ions are electrophilic and might compete with carcinogens for binding sites on DNA. Also, several experiments suggest that intracellular deficiency of magnesium contributes to cancer (9,35). Epithelial cell proliferation is increased in the gastro-intestinal tracts including colon with increased susceptibility to cancer before the appearance of tumors (36,37). Recently, it is reported that dietary calcium decreases hyperproliferation of colonic epithelial cells in human subjects at high risk for colon cancer (37,38) and the incidence of carcinogen-induced colonic tumors in rodent (39). This effect of calcium might be due to a specific effect on the complex events controlling cycling of intestinal mucosal cells (40). Another metal ion, magnesium has an effect on cell cycle and is recognized as a possible central regulator of cell proliferation and growth (11). Thus, magnesium could prevent cell division of intestinal mucosal cells with hyperproliferation (11,41). These cell-biological functions of magnesium may be related to the protective effect of the agent on MAM acetate-induced carcinogenesis. However, at present, as in the case of calcium, the effect of magnesium on the cell cycle is most considerable for the mechanism by which the agent affects MAM acetate-induced tumorigenesis. The question also arises why the inhibition was not dose-related and was more effective at the lower dose. We do not have any clear reason for this. In summary, it was demonstrated that magnesium hydroxide is a potent inhibitor in MAM acetate-induced carcinogenesis in rats. Further experiments are underway in our laboratory to elucidate the mechanisms as well as explore the dose-relation of the anticarcinogenic effect of magnesium hydroxide.

T.Tamka et al.

Received on August 31, 1988; revised on November 21, 1988; accepted on November 23, 1988

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