The Journal of Nutrition Vol. 128 No. 3 March 1998, pp. 536-540

Dietary Maltitol Decreases the Incidence of 1,2-Dimethylhydrazine-Induced Cecum and Proximal Colon Tumors in Rats^1,2

Midoriko Tsukamura^*, Hidemi Goto^*, Tomiyasu Arisawa^*, Tetsuo Hayakawa^*, Naoya Nakai, Taro Murakami, Noriaki Fujitsuka, and Yoshiharu Shimomura^{, 3}

^* Department of Internal Medicine II, School of Medicine, Nagoya University, Showa-ku, Nagoya, 466, Japan and  Department of Bioscience, Nagoya Institute of Technology, Showa-ku, Nagoya, 466, Japan

	ABSTRACT

Abstract Introduction Methods Results Discussion References

Maltitol is fermented in the colon due to only partial hydrolysis in the small intestine. In the present study, we examined effects of dietary maltitol on dimethylhydrazine-induced intestinal tumor in rats. In experiment 1, rats were fed a fiber-free diet or diets supplemented with 1 or 5 g/100 g maltitol for 27 wk. Each group of rats was injected with dimethylhydrazine or vehicle alone for the first 14 wk of the experimental period. Maltitol supplementation at 1 g/100 g of the diet significantly reduced tumor incidence in the cecum and the 5% supplement reduced tumor incidence in both the cecum and proximal colon in dimethylhydrazine-treated rats. In experiment 2, we investigated the effect of the 1 g/100 g maltitol diet on the short chain fatty acid concentrations in cecal contents of placebo and dimethylhydrazine-treated rats. Intake of the 1 g/100 g maltitol diet doubled (P < 0.05) the concentration of butyrate but did not affect acetate or propionate in the cecal contents. These results suggest that dietary maltitol has a protective effect against dimethylhydrazine-induced tumors in rat cecum and proximal colon and that butyrate produced by bacterial fermentation of maltitol in the cecum may be involved in the protection.

	INTRODUCTION

Abstract Introduction Methods Results Discussion References

Fermentation by anaerobic bacteria to break down dietary and other substrates in the large intestine is an important component of normal activity of the organ to obtain energy for growth and maintenance of cellular functions. Fermentation is mainly dependent upon the amount and type of substrate available to the microflora because the substrates will determine the magnitude and rate of metabolic events in the colon (Cummings and Englyst 1987). Some foods are considered to be indigestible (or poorly digestible). These include dietary fibers, oligosaccharides, some starches and sugar alcohols, some of which are fermented in the colon (Cummings and Englyst 1987). The end products of the fermentation process are short chain fatty acids (principally acetate, propionate and butyrate) and gases (carbon dioxide, methane and hydrogen). Butyrate is the preferred fuel of colonic epithelial cells (Roedgier 1982), slows the growth of cultured colon carcinoma cells and promotes expression of differentiation markers (Gum et al. 1987, Heruth et al. 1993, Kim et al. 1980).

Many investigators have examined the effect of dietary fibers on carcinogen-induced colon cancer in rats and reported that some insoluble dietary fibers such as wheat bran show a protective effect (McIntyre et al. 1993), whereas some soluble fibers such as pectin may enhance colon carcinogenesis (Bauer et al. 1979). Both wheat bran and pectin are fermentable in rat colon (particularly in the cecum). The latter especially is highly fermented. Reasons for the difference in these fibers' effects on carcinogenesis are not known, but more butyrate is produced from wheat bran than from pectin by fermentation in the colon (Lupton and Kurtz 1993).

Besides dietary fibers, resistant starch is also fermentable in the colon and decreases colonic mucosal proliferation (van Munster et al. 1994). Lactulose has a protective effect against colon cancer (Samelson et al. 1985), perhaps in association with the lowering of the pH of colonic lumen (van Berge Henegouwen et al. 1987).

Maltitol (-D-glucopyranosyl-1,4-sorbitol) is a sugar alcohol produced by the hydrogenation of maltose (Suzuki and Tamura 1988). This sugar alcohol is hydrolyzed by small intestinal disaccharidases much more slowly than maltose (Yoshizawa et al. 1975). Therefore, when maltitol is ingested with a meal, the majority of dietary maltitol likely reaches the large intestine and then is fermented by microflora (Oku et al. 1991).

Because maltitol has become a popular sugar substitute in Japan (Suzuki and Tamura 1988), it is important to determine whether dietary maltitol suppresses or promotes colon carcinogenesis. In the present study, we examined effects of maltitol on the incidence of 1,2-dimethylhydrazine (DMH)-induced colon cancer in rats.

	MATERIALS AND METHODS

Abstract Introduction Methods Results Discussion References

This study was performed according to the guidelines for animal experimentation set by Nagoya University.

Experiment 1

Animals and diets.  Eighty-nine male F344 rats (4 wk old) (CLEA Japan, Tokyo) were used. The rats were housed three per cage in suspended wire-bottomed cages in an animal room with constant temperature (23°C) and a 12-h light-dark cycle. Fresh air was supplied constantly to the animal room, and a ventilation system for the room was designed to exchange all of the air in the room with fresh air every 4.6 min. The rats consumed a nonpurified commercial diet (CE2; CLEA Japan, Tokyo) for 1 wk and then were assigned randomly to three dietary groups: control (fiber-free) diet group, and 1 and 5% maltitol diet groups. The experimental diets were semipurified powder diets and were prepared to our specifications by CLEA Japan. The composition of each experimental diet is shown in Table 1. Rats were given free access to the appropriate experimental diet and tap water for 27 wk. One week after giving the experimental diet, each group of rats was subdivided into two groups: a DMH-treated group and a placebo group. Rats in the DMH-treated group were injected subcutaneously once each week with DMH (Aldrich Chemical, Milwaukee, WI) dissolved in 0.2 mol/L sodium bicarbonate buffer (pH ~8) with 1 mmol/L EDTA at a dosage of 20 mg/kg body weight for 14 wk (from week 2 through week 15). A fresh DMH solution was prepared immediately before use. Rats in the placebo group were injected with an equal volume of the buffer without DMH as described above.

Measurements of body weight and food intake.  Rat body weight was measured weekly, and food intake was measured during the experiment at weeks 8, 17 and 26.

Measurements of pH of cecum and colon contents.  On the final day of the experiment, rats were killed by cervical dislocation. A laparotomy was performed rapidly through a midline incision exposing the large intestine. The pH of the intestinal contents was measured using a glass electrode (needle combination pH probe) with a digital pH meter (Toa HM-20E pH meter, Toa, Tokyo), which was inserted into a 5-mm incision of the bowel wall in the cecum and 5 cm distal to the cecal-proximal colon junction.

Autopsy.  The cecum and colon were removed, cut open, flushed with ice cold saline and weighed. The presence or absence of tumors was noted as was their relative position that is, either in cecum, proximal colon or distal colon. The tumor size was measured in three dimensions (at an angle of 90°), and a tumor size index (mm³) as a measure of mass was calculated for each rat by multiplying these dimensions. The majority of tumors were identified readily. When the identity of tumors was not clear, the tissue was examined histologically after staining with hematoxylin and eosin (Ward 1974). Tumor incidence data represent the combined results for adenomas and adenocarcinomas.

Experiment 2

Animals and diets.  Forty-three male F344 rats (4 wk old) were used and were housed under the same conditions as described in experiment 1. After giving the CE2 nonpurified diet for 1 wk, rats were divided randomly into two diet groups: control (fiber-free) diet group and 1% maltitol diet group. The rats were fed the appropriate diet for 9 wk and were allowed free access to tap water throughout the experiment. One week after giving the experimental diet, each group of rats was subdivided into two groups: DMH-treated group and placebo group. Rats were treated with DMH or placebo for 8 wk as described in experiment 1.

Fecal samples, food intake and body weights.  Rats were individually caged, and average fecal wet weight/24 h was measured by collecting all feces, separate from urine, from cages during three serial 24-h periods in the final week of the experiment. The feces were frozen at 80°C and were used for measurement of fecal dry weight by the method of Lupton and Kurtz (1993). During the period of fecal collection, food intake of each rat also was measured. Body weight was measured weekly.

Cecum contents.  On the final day of the experiment, rats were killed as described in experiment 1, and cecum contents were collected and stored at 80°C.

Analyses of short-chain fatty acids.  Short-chain fatty acids in cecal contents were measured by the gas-liquid chromatographic method of McIntyre et al. (1993).

Statistical analysis.  Values are means ± SD. To evaluate the differences among groups, data other than tumor incidence were analyzed by two-way analysis of variance (diet × DMH treatment). If a significant difference was found, Scheff's test was employed (Ott 1993). Values with P < 0.05 were considered significant. Tumor incidence or tumor numbers were analyzed by the chi-square test (P < 0.05). Statistical analyses were performed using Stat View 4.0 (Abacus Concepts, Berkeley, CA).

	RESULTS

Abstract Introduction Methods Results Discussion References

Experiment 1

Food intake and weights of body, cecum and colon.  Food intake measured during weeks 8, 17 and 26 in the experiment was not affected by supplementation of maltitol in the diet or DMH treatment (Table 2). Body weight on the final day of the experiment was lower in DMH-treated rats than in those administered placebo. Weights of cecum and colon were not affected by the DMH treatment or the maltitol diet (Table 2).

pH of large intestinal contents.  The pH of cecal contents in rats fed maltitol was significantly lower than in the control (P < 0.05), except that the pH decrease due to the 1 g/100 g maltitol diet was not significant in the DMH treated rats (Table 3). DMH treatment did not affect cecal pH, and the pH of colon contents was not affected by DMH treatment or the maltitol diet (Table 3).

Tumor incidence.  Tumors were not observed in the large intestine of rats treated with placebo in any dietary group. Tumor incidence in rats treated with DMH was significantly lower in those fed 1% maltitol diet group than in those fed the control diet (Table 3, P < 0.05). The same trend was observed in the 5% maltitol diet group (P = 0.072; Table 4). Tumor numbers and sizes (data not shown) in the tumor-bearing rats were not different among the three dietary groups (Table 4).

Tumor incidence in the different sections of the large intestine (cecum, proximal colon and distal colon) is shown in Table 5. Twenty percent of rats in the control group had a cecal tumor, but no rats in either maltitol diet group had tumors. In the proximal colon, 45% of rats in the control diet group had tumors, and the tumor incidence in this tissue was significantly lower in the 5% maltitol diet group (P < 0.05) and tended to be lower in the 1% maltitol diet group (P = 0.091). On the other hand, the tumor incidence in the distal colon was 30% for rats in the control diet group and was not different among the three dietary groups, indicating that dietary maltitol did not affect tumor incidence in the distal colon. Tumor numbers and sizes (data not shown) in the tumor-bearing rats for both proximal and distal colon were not different among the three dietary groups.

Experiment 2

Food intake, body weight and fecal dry weight.  Food intake was not significantly affected by the maltitol diet or DMH treatment, but in the control groups, it tended to be less in rats treated with DMH than in those treated with placebo (P = 0.071; Table 6). Body weight in the control groups was significantly less in rats treated with DMH than in those treated with placebo, but in the maltitol diet groups, there was no difference between placebo- and DMH-treated rats (Table 6). There were no significant differences in the food intakes or body weights due to diet (Table 6) as observed in experiment 1. 

Fecal dry weight of rats did not different among the groups (Table 6).

Short-chain fatty acids in cecum contents.  The major short-chain fatty acids in cecum contents were acetate, propionate and butyrate. Concentrations of acetate and propionate were not different among the groups (Table 7). Butyrate concentration in the contents of rats fed 1 g/100 g maltitol was twice that of controls (P < 0.05).

	DISCUSSION

Abstract Introduction Methods Results Discussion References

The present study examined the effect of dietary maltitol on colon cancer induced by DMH treatment in rats. Experiment 1 in this study showed that 1 g/100 g (P < 0.05) and 5 g/100 g (P = 0.072) supplementation of maltitol in the experimental diet decreased tumor incidence in the whole large intestine. When the tumor incidence was analyzed on different sections of the large intestine, a significant effect was observed in the cecum for the 1% maltitol diet group and in both the cecum and the proximal colon for the 5% maltitol diet group but not in the distal colon. The maltitol supplementation decreased the pH of the cecal contents but not the pH of the content in the colon, indicating that the maltitol ingested was fermented exclusively in the cecum as reported previously (Oku et al. 1991). These results suggest that the fermentation of maltitol is responsible for the protective effect against colon cancer.

It has been reported that indigestible components of food produce different amounts and compositions of short-chain fatty acids when they are fermented (Lupton and Kurtz 1993, McIntyre et al. 1993). Therefore we measured the fatty acids in the cecal contents. Among the fatty acids, butyrate is especially interesting because it has been reported to be an important energy source for normal colonocytes (Roediger 1982), to slow the growth of cultured colon carcinoma cells and to promote expression of differentiation markers (Gum et al. 1987, Heruth et al. 1993, Kim et al. 1980). Furthermore, the fecal butyrate concentration of patients with colorectal cancer is lower than those of healthy controls (Weaver et al. 1988). In the present study, acetate, propionate and butyrate were found in the cecum contents, and only the butyrate concentration was increased by supplementation of 1 g/100 g maltitol in the diet, suggesting that butyrate may play an important role in the protective effect of dietary maltitol against DMH-induced carcinogenesis in rat cecum and proximal colon. McIntyre et al. (1991) have reported that the pH of colonic contents gradually rises during the transport from cecum to sigmoid colon, probably due to the absorption of short-chain fatty acids. They also reported that oat bran and guar gum are highly fermented in the proximal colon but do not influence the distal luminal environment because short-chain fatty acids are absorbed rapidly (McIntyre et al. 1991). Maltitol may have the same effect as these fibers; therefore, the maltitol did not affect the distal colon. McIntyre et al. (1991) proposed that the best way to maintain relatively high butyrate concentrations as well as low pH in the distal colon, the major site for colon cancer (Schottenfeld and Haas 1978), may be by feeding a fiber with medium fermentability, such as wheat bran.

The degree of fermentation is dependent upon the physical nature of the fiber (Cummings 1981), the surface area exposed to bacteria (Hsu and Penner 1989) and duration of the exposure (Stephen et al. 1987). Highly fermentable fibers such as guar gum and pectin enhance tissue hypertrophy and carcinogenesis (Jacob and Lupton 1986). It has been reported that fermentation of dietary guar gum and pectin increased the concentrations of short-chain fatty acids, especially acetate and propionate, in the contents of the large intestine. These fatty acids were reported to be a primary factor in lowering the pH in the cecum contents, being associated with promotion of cell proliferation (Jacob and Lupton 1986, Lupton and Kurtz 1993). Because butyrate production by fermentation was induced by guar gum, but not pectin, cecum hypertrophy does not appear to be related to the increase in butyrate (Lupton and Kurtz 1993, McIntyre et al. 1993). Because acetate and propionate are the major short-chain fatty acids produced by fermentation, these fatty acids were reported to be important in lowering the pH in the cecum contents; these effects of fatty acids are associated with promotion of cell proliferation (Jacob and Lupton 1986, Lupton and Kurtz 1993). It has been reported that an increase in cell proliferation in the presence of a carcinogen may enhance colon tumorigenesis (Newmark and Lupton 1990). It is possible that fermentation vigorous enough to produce hypertrophy may increase the risk of colon cancer. On the other hand, hypertrophy of the cecum was not observed in this study using 1 and 5 g/100 g maltitol diets. This may be due to the relatively small amounts of maltitol supplemented in the diet, because 10 g/100 g maltitol supplementation has been reported to cause hypertrophy of the cecum (Oku et al. 1983). It remains to be clarified whether the amount of maltitol supplementation that causes hypertrophy of the cecum would have a protective effect against colon cancer.

In conclusion, the present study demonstrated that dietary maltitol has a protective effect against carcinogenesis in the cecum and the proximal colon induced by DMH treatment in rats, and butyrate produced by bacterial fermentation of maltitol in the cecum may be involved in the protective effect. Many sugar alcohols are used industrially as sugar substitutes. However, it has not been reported whether sugar alcohols affect colon carcinogenesis. This is the first report to show the protective effect of maltitol against colon cancer. Other sugar alcohols may have the same effect on colon cancer, but this remains to be examined. Butyrate production by bacterial fermentation in the cecum might be a useful index for the protective effect of sugar alcohols against colon cancer, and therefore it may be important to find sugar alcohols which increase the butyrate concentration throughout the large intestine.

	ACKNOWLEDGMENTS

We would like to express our gratitude to Michael V. Bodman, language consultant of our department, for giving us suggestions on language.

	FOOTNOTES

Manuscript received 30 December 1996. Initial reviews completed 3 February 1997. Revision accepted 1 December 1997.

	LITERATURE CITED

Abstract Introduction Methods Results Discussion References

• American Institute of Nutrition Report of the American Institute of Nutrition ad hoc committee on standards for nutritional studies. J. Nutr. 1977; 107:1340-1348
• Bauer H. G., Asp N. G., Öste R., Dahlqvist A., Fredlund P. E. Effect of dietary fiber on the induction of colorectal tumors and fecal -glucuronidase activity in the rat. Cancer Res. 1979; 39:3752-3756 [Abstract/Free Full Text]
• Cummings J. H. Dietary fiber. Br. Med. Bull. 1981; 37:65-70 [Free Full Text]
• Cummings J. H., Englyst H. N. Fermentation in the human large intestine and the available substrates. Am. J. Clin. Nutr. 1987; 45:1243-1255 [Free Full Text]
• Gum J. R., Kam W. K., Byrd J. C., Hicks J. W., Sleisenger M. H., Kim Y. S. Effects of sodium butyrate on human colonic adenocarcinoma cells. J. Biol. Chem. 1987; 262:1092-1097 [Abstract/Free Full Text]
• Heruth D. P., Zirnstein G. W., Bradley J. F., Rothberg P. G. Sodium butyrate causes an increase in the block to transcriptional elongation in the c-myc gene in SW837 rectal carcinoma cells. J. Biol. Chem. 1993; 268:20466-20472 [Abstract/Free Full Text]
• Hsu J. C., Penner M. H. Influence of cellulose structure on its digestibility in rat. J. Nutr. 1989; 119:872-878
• Jacob L. R., Lupton J. R. Relationship between colonic luminal pH, cell proliferation, and colon carcinogenesis in 1,2-dimethylhydrazine treated rats fed high fiber diet. Cancer Res. 1986; 46:1727-1734 ^{[Abstract/Free Full Text]}
• Kim Y. S., Tsao D., Siddiqui B., Whitehead J. S., Arnstein P., Bennet J., Hicks J. Effects of sodium butyrate and dimethylsulfoxide on biochemical properties of human colon cancer cells. Cancer 1980; 45:1185-1192 [Medline]
• Lupton J. R., Kurtz P. P. Relationship of colonic luminal short-chain fatty acids and pH to in vivo cell proliferation in rats. J. Nutr. 1993; 123:1522-1530
• McIntyre A., Gibson P. R., Young G. P. Butyrate production from dietary fiber and protection against large bowel cancer in a rat model. Gut 1993; 34:386-391 [Abstract/Free Full Text]
• McIntyre A., Young G. P., Taranto T., Gibson P. R., Ward P. B. Different fibers have different regional effects on luminal contents of rat colon. Gastroenterology 1991; 101:1274-1281 [Medline]
• Newmark H. L., Lupton J. R. Determinants and consequences of colonic luminal pH: implications for colon cancer. Nutr. Cancer 1990; 14:161-173 [Medline]
• Oku T., Akiba M., Lee M. H., Moon S. J., Hosoya N. Metabolic fate of ingested [¹⁴C]-maltitol in man. J. Nutr. Sci. Vitaminol. 1991; 37:529-544
• Oku, T., Konishi, F., Kanda, A. & Hosoya, N. (1983) Biochemical and morphological changes of gastrointestinal tract by nondigestible carbohydrate. In: Dietary FiberIts Food Scientific and Nutritional Approach (Inami, S., ed.), pp. 75-103. Shinohara Publisher, Tokyo.
• Ott, R. L. (1993) Multiple comparisons. In: An Introduction to Statistical Method and Data Analysis (Lozyniak, K. & Krikorian, S., eds.), 4th ed., pp. 807-841, Duxbury Press, Belmont, CA.
• Roediger W.E.W. Utilization of nutrients by isolated epithelial cells of the rat colon. Gastroenterology 1982; 83:424-429 ^[Medline]
• Samelson S. L., Nelson R. L., Nyhus L. M. Protective role of fecal pH in experimental colon carcinogenesis. J. R. Soc. Med. 1985; 78:230-233 [Abstract]
• Schottenfeld, D. & Haas, J. F. (1978) Epidemiology of colon cancer. In: Gastrointestinal Tract Cancer (Lipin, M. & Good, R. A., eds.), pp. 207-240. Plenum, New York.
• Stephen A. M., Wiggins H. S., Cummings J. H. Effect of changing transit time on colonic microbial metabolism in man. Gut 1987; 28:601-609 [Abstract/Free Full Text]
• Suzuki, M. & Tamura, T. (1988) Sweeteners: low-energetic and low- insulinogenic. In: Diet and Obesity (Bray, G. A., Leblanc, J., Inoue, S. & Suzuki, M., eds.), pp. 163-173. Japan Sci. Soc. Press Tokyo/S., Karger Basel.
• Tulung B., Remesy C., Demigne C. Specific effects of gur gum or gum arabic on adaptation of cecal digestion to high fiber diets in the rat. J. Nutr. 1987; 117:1556-1561
• van Berge Henegouwen, G. P., van der Werf S.D.J., Ruben A. T. Effect of long term lactulose ingestion on secondary bile salt metabolism in man: potential protective effect of lactulose in colonic carcinogenesis. Gut 1987; 28:675-680 ^{[Abstract/Free Full Text]}
• van Munster I. P., Tangerman A., Nagengast F. M. Effect of resistant starch on colonic fermentation, bile acid metabolism, and mucosal proliferation. Dig. Dis. Sci. 1994; 39:834-842 [Medline]
• Yoshizawa S., Moriuchi S., Hosoya N. The effects of maltitol on rat intestinal disaccharidases. J. Nutr. Sci. Vitaminol. 1975; 21:31-37
• Ward J. M. Morphogenesis of chemically induced neoplasms of the colon and small intestine in rats. Lab. Invest. 1974; 30:505-513 [Medline]
• Weaver G. A., Krause J. A., Miller T. L., Wolin M. J. Short chain fatty acid distributions of enema samples from a sigmoidoscopy population: an association of high acetate and low butyrate ratios with adenomatous polyps and colon cancer. Gut 1988; 29:1539-1543 [Abstract/Free Full Text]

0022-3166/98 $3.00 ©1998 American Society for Nutritional Sciences