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Long‐term effect of bifidobacteria and Neosugar on precursor lesions of colonic cancer in cf1 mice a
Malcolm Koo & A. Venketeshwer Rao
a
a
Department of Nutritional Sciences, Faculty of Medicine, University of Toronto, 150 College St., Toronto, Ontario, M58 1A8, Canada Version of record first published: 04 Aug 2009.
To cite this article: Malcolm Koo & A. Venketeshwer Rao (1991): Long‐term effect of bifidobacteria and Neosugar on precursor lesions of colonic cancer in cf1 mice, Nutrition and Cancer, 16:3-4, 249-257 To link to this article: http://dx.doi.org/10.1080/01635589109514163
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Long-Term Effect of Bifidobacteria and Neosugar on Precursor Lesions of Colonic Cancer in CF1 Mice Downloaded by [Universite Laval] at 22:50 19 April 2013
Malcolm Koo and A. Venketeshwer Rao
Abstract This investigation was undertaken to study the role of Bifidobacteria and bifidogenic factor Neosugar in the process of 1,2-dimethylhydrazine-induced colonic carcinogenesis in CF1 mice. Intestinal colonization and selective proliferation of Bifidobacteria were achieved by oral administration of indigenous Bifidobacteria and the incorporation of 5% Neosugar in the diet of animals. The Bifidobacteria were isolated from the feces of CF, mice and were identified to be Bifidobacterium pseudolongum biovar b. This incidence of aberrant crypts andfoci were significantly lower 38 weeks after the last injection of the carcinogen in animals fed Bifidobacteria than in animals treated with the carcinogen alone. The aberrance also appeared to be confined to the more distal end of the colon in animals fed bifidogenic diet. Such changes in the precursor lesions of colonic carcinogenesis are presumably due to the increase in the number of Bifidobacteria and their acidifying action in the lower intestinal tract of the animals. (Nutr Cancer 16, 249-257, 1991)
Introduction
Intestinal bacteria play an important role in the process of colonic carcinogenesis. Major evidence supporting this view comes from observations of lower incidence of chemically induced colon cancer in germ-free animals (1,2) and the ability of intestinal bacteria to produce various possible carcinogens and promoters (3). With respect to enhanced cancer risk, secondary bile acids (4) and cholesterol metabolites (5) received the greatest attention. Not all intestinal bacteria, however, are associated with the etiology of colon cancer. Lactobacilli have been reported as being protective against chemically induced colonic carcinogenesis in rats (6). Similarly, Bifidobacteria, which are also acid-producing bacteria, have been shown to be protective against colonic carcinogenesis (7). It was reported that feeding of naturally fermented sour milk but not artificially acidified milk in the diet significantly reduced the number of colonic tumors induced by dimethylhydrazine in rats (8). The authors are affiliated with the Department of Nutritional Sciences, Faculty of Medicine, University of Toronto, Toronto, Ontario M58 1A8, Canada.
Copyright © 1991, Lawrence Erlbaum Associates, Inc.
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In the same study, a parallel increase in fecal bifidobacterial count was also observed. In addition to the intraluminal acidification effect, possible antibacterial activity of Bifidobacteria might be responsible for the antitumorigenic effect of sour milk. Bifidobacteria are Gram-positive anaerobic bacteria. They constitute a major part of the normal human intestinal and fecal flora throughout life. They appear in the stools two to five days after birth and rapidly establish themselves to be the most predominant bacterial group in the colon, ranging from 109 to 1011 cells/g feces. However, their number decreases significantly during senescence (9,10). Bifidobacteria are unique in their metabolism of carbohydrates. They produce acetic and L-(+)-lactic acids in a molar ratio of 3:2 from glucose through the characteristic fructose 6-phosphate shunt (11). To enhance the selective proliferation and colonization of Bifidobacteria in the intestinal tract, a bifidogenic factor, Neosugar, was incorporated into the diet of animals receiving Bifidobacteria orally (12). Neosugar is a mixture of fructooligosaccharides that is not digested by gastrointestinal enzymes of humans or animals (13) but readily utilized by certain intestinal bacteria including Bifidobacteria, Bacteroides fragilis, Peptostreptococcus spp, and Klebsiella pneumoniae. Acetic, propionic, and butyric acids are produced by these bacteria, which may substantially reduce the intestinal pH (14). Neosugar, on the other hand, is not used by other undesirable bacteria such as Escherichia coli, Clostridium perfringes, and C. paraputrificum (12). The principal objective of this study was to investigate the effect of a bifidogenic regimen consisting of oral administration of viable Bifidobacteria and dietary supplementation of Neosugar on 1,2-dimethylhydrazine-induced colonic carcinogenesis in CFj mice. Aberrant crypt in the colon was used to assess the carcinogenic process. When two or more aberrant crypts are found adjacent to each other, the resulting cluster is termed aberrant foci. McLellan and Bird (17) showed that a single crypt may be replaced by multicrypt foci from two to four weeks after the final injection. They suggested that multicrypt foci may be associated with higher doses of carcinogen. However, the exact relationship between single-crypt and multicrypt foci is not clear. Both parameters were determined in the present study. Several lines of evidence indicate that aberrant crypts represent precursor lesions of chemically induced colon cancer (15). Aberrant crypts exhibit many common characteristics of precursor lesions. They are reported to be specifically induced by colon carcinogens, and the induction has been reported to be dose dependent. The frequency of aberrant crypts is modulated by agents that modify the carcinogenic process, often with the degree observed when colonic tumors are the end points. In addition, an increased proliferation rate, which is common to early changes in colon carcinogenesis, is observed in aberrant crypts. Methods Experimental Design Three experimental groups were established: mice treated with 1,2-dimethylhydrazine (DMH) and fed Bifidobacteria and a bifidogenic diet (« = 20), mice treated with DMH and fed a normal diet {n = 21), and control mice that did not receive DMH and were fed a normal diet (n = 5). The normal diet used was AIN-76A (ICN Biomedicals, Cleveland, OH), and 5% Neosugar P (Meiji Seika Kaisha, Tokyo, Japan) was added to the AIN-76A diet to form the bifidogenic diet. Female CFj mice (Charles River, Montreal, Quebec) weighing 21.4 ± 1.5 g were used in the study. They were placed on their respective diets three days after arrival. After a two-week acclimation period, treatment mice were given the first of the six weekly subcutaneous injections of DMH solution (20 mg/kg body wt; Aldrich Chemical, Milwaukee, WI). The DMH solution was freshly made each time before injection in 0.001 M
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ethylenediaminetetraacetic acid (EDTA, Sigma Chemical, St. Louis, MO) solution and brought to pH 6.5 with sodium bicarbonate. Control animals were injected likewise with EDTA solution. One week after the last injection, oral intubation of viable bifidobacterial suspension (lOVmouse) twice a week was started. Animals were killed at three time points: 18, 28, and 38 weeks after the last carcinogen injection. Ten animals, four from each treatment group and two from the control, were killed at Week 18 by cervical dislocation. Colons were fixed in 10% phosphate-buffered formalin and quantified for the presence of aberrant crypts. Ceca along with their content were analyzed for their pH and acetic acid content. Seven mice, three from each treatment group and one from the control, were killed at Week 28 for aberrant crypt determination. The remaining animals were killed 38 weeks after the last injection of the carcinogen. Overnight fecal samples were collected for the enumeration of total aerobes, total anaerobes, and Bifidobacteria. Ceca and their content were analyzed for acetic acid content, and colons were analyzed for the presence of aberrant crypt.
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Bacteriological Preparation Bifidobacterial suspension used in the oral intubation was isolated from the feces of CV1 mice. Overnight fecal sample was collected, homogenized in peptone water (GIBCO Laboratories Life Technologies, Madison, WI), and streaked on blood-base glucose blood liver agar for selective isolation of Bifidobacteria. The composition of the blood-base glucose blood liver agar is shown in Table 1. The inoculated plates were incubated in anaerobic jars at 37°C for 72 hours. The anaerobic requirement of Bifidobacteria was satisfied by the use of Anaerocult C Gas Pak (Merck, Darmstadt, Germany). After incubation, all distinct types of colonies were examined under a microscope and subjected to further biochemical analysis and the fructose 6-phosphate phosphoketolase enzymatic confirmation of Bifidobacteria (16). Identification of Bifidobacteria at the species level was achieved by matching the carbohydrate utilization Table 1. Composition of Glucose Blood Liver Agar" Ingredient Meat extract Proteose peptone Peptone Soy bean peptone Yeast extract Liver extract powder Glucose Soluble starch K 2 HPO 4 KH2PO4 MgSO4-7H2O NaCl MnSO4 Defoaming agent Polysorbate 80 L-Cysteine hydrochloride Agar Blood agar base
Composition* 2.4 10.0 3.0 3.0 5.0 3.2 10.0 0.5 1.0 1.0 0.01 0.01 0.007 0.2 1.0 0.3 15.0 40.0
a: Formula based on Mitsuoka (35) with slight modifications. b: Expressed in grams per liter of water.
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profile of isolated Bifidobacteria with that in the literature (16) by means of the API system (API Laboratory Products, St. Laurent, Quebec). Stock culture of the isolated Bifidobacteria was maintained on Lactobacillus MRS agar. The agar was prepared by adding 55 g of Lactobacillus MRS broth (Difco Laboratories, Detroit, MI) and 15 g of agar (GIBCO Laboratories Life Technologies) to 1 liter of distilled water. Cysteine hydrochloride (Sigma Chemical) was added to the medium after it was autoclaved to a final concentration of 0.03%. The cell suspension used in oral intubation was made by suspending saline-washed 48-hour culture broth in phosphate-buffered saline (pH 6.5) to a concentration of at least 109 viable cells per milliliter. Bacterial Enumeration
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Samples were homogenated and serially diluted with peptone water for plating in a standard fashion. Plate count agar (Difco Laboratories) was used for enumeration of total aerobic bacteria, and reinforced clostridial agar (Difco Laboratories) was used for total anaerobic bacterial count. Bifidobacteria were enumerated in Lactobacillus MRS agar. All plates were incubated at 37°C for 48 hours in appropriate atmospheric conditions. Laboratory Procedures Cecal samples were homogenized using a vortex mixer with nine volumes of physiological saline in capped polypropylene centrifuge tubes. The air in the tubes was replaced by nitrogen. The homogenates were then centrifuged at 3,000 rpm (=1,650 g) for 15 minutes. The pH of the supernatants was measured immediately. Acetic acid concentration of the cecal samples was quantified by the enzymatic ultraviolet method (Boehringer Mannheim Canada, Dorval, Quebec). Aberrant crypts were scored using the procedure described by McLellan and Bird (17). Colonic samples were stained with methylene blue dye (1% in phosphate-buffered saline) for 10 minutes. The mucosal surfaces were examined under X100 magnification with a light microscope. To determine the distribution of aberrant crypts, colonic mucosa 5 cm anterior to the anus was divided into three sections where R represented the first 2 cm from the rectal end, M was the next 2 cm, and C was the 1st cm from M. Criteria used for identification of aberrant crypts were elongation and increased size of crypt cells, thickening of epithelial lining, and increased pericryptal zone (18). Statistical Methods All results were subjected to Student's / test by means of the SAS statistical package (SAS Institute, Cary, NC). The Satterthwaite approximation was used for samples with unequal variances. All results were expressed as means ± SD. Statistical comparisons of bacterial counts were performed on logarithmic-transformed raw data, and the results were expressed as logarithm to the base 10 of the number of viable bacteria per gram of feces. Results No aberrant crypts were identified in the colons of control animals at all three times of termination. One adenocarcinoma was found in one of the mice in the carcinogen-treated group 28 weeks after the last carcinogen injection; otherwise, all animals were in good general condition. Deschner and Long (19), using identical carcinogen, animals, and injection protocol, reported that 83% of the animals were tumor bearing, with a frequency of 2.1 macroscopically visible colonic tumors per mouse between the 20th and 45th week of the experiment. The extremely low tumor incidence observed in the present study is somewhat surprising. A higher tumor incidence may be seen if the experiment had continued
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for another two to three months. DMH was the choice of carcinogen because it induces lesions mainly in the distal colon and follows the adenomatous polyps-adenocarcinoma sequence similar to that observed in humans. The number of injections was kept to a minimum to avoid the carpeting phenomenon of colonic tumors, which may otherwise overwhelm any protective effect contributed by Bifidobacteria. Taken together, the mean numbers of aberrant crypts and aberrant foci were lower in animals receiving the bifidogenic diet. The incidence of occurrence was 100% in both treatment groups. Initially, at Week 18, the mean number of aberrant crypts was significantly lower in animals fed the bifidogenic diet. At Week 18 the mean number of aberrant foci was statistically lower in animals fed the bifidogenic diet, and at Week 38 both parameters were significantly lower in these animals. The trends of the mean aberrant crypts and foci are shown in Figures 1 and 2, respectively. There appears to be a diverging trend between the two groups of animals in terms of the incidence of both aberrant crypts and foci. The distribution of aberrant foci is shown in Figure 3. A similar pattern of distribution was also observed in aberrant crypts. The majority of the aberrant foci were found at the mucosa of the midcolon. Starting from Week 28, aberrant foci began to appear in the cecal mucosa exclusively in the mice treated with DMH alone. At Week 38, the mean bifidobacterial count in the feces of animals fed bifidogenic diet was increased from 8.85 ± 0.20 to 9.45 ± 0.19 (p < 0.05). The logarithmic ratio of fecal anaerobes to aerobes was also elevated from 0.20 ± 0.01 to 0.92 ± 0.34 (p < 0.05). Mean cecal weight, pH, and acetic acid content of the animals are summarized in Table 2. Mean full cecal weights of animals fed bifidogenic diet were significantly (p < 0.05) higher in Weeks 18 and 38. Cecal pH was significantly lower in these animals (p < 0.001) at Week 18, but the reduction was not significant at Week 38. Although cecal acetic acid concentration in bifidogenic diet-fed animals was almost twice that of the other group, the changes were not statistically significant.
40 35 C
30
O O
25
>
20
6 -
15 10
Figure 1. Effect of bifidogenic diet on nos. of aberrant crypts in 1,2-dimethylhydrazine (DMH)-treated CF, mice. Horizontal axis, weeks after last injection of DMH. Symbols are as follows: a, p < 0.05; b,/> < 0.01.
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0*
10
15 20 25 30 35 40 Time (week)
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10
15
20
35 40
25
Time (week) 100%
Figure 2. Effect of bifidogenic diet on nos. of aberrant foci in 1,2-dimethylhydrazine (DMH)-treated CF, mice. Horizontal axis, weeks after last injection of DMH. Symbols are as follows: a, p < 0.05; b , p < 0.01.
n n
Distribution of Aberrant Foci
75%-
50%-
25% - !
Bifido
DMH
Bifido
Week 18 t
) Rectal mucosa
• •
Cecal mucosa
DMH
Week 28 I
Bifido
DMH
Week 3 8
.1 Midcolon mucosa
Figure 3. Effect of bifidogenic diet on distribution of aberrant foci in 1,2-dimethylhydrazine (DMH)-treated CF, mice. Horizontal axis, weeks after last injection of DHM in animals fed bifidogenic and normal diet. Aberrance was confined to more distal end of colon in mice fed bifidogenic diet at all 3 time points.
Discussion
Feeding of indigenous Bifidobacteria was effective in significantly increasing their number in the lower intestinal tract of the animals. Indigenous strains of Bifidobacteria present in the feces of the animals are presumably those that have already possessed or acquired desirable characteristics for a successful colonization in the intestine of their host. The matching of host specificity may therefore facilitate a rapid proliferation of Bifidobacteria when they are
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Table 2. Effect of Bifidogenic Diet on Full Cecal Weight, Cecal. pH , and Acetic Acid in DMH-Treated CFj Mice"'" DMH Treated Normal diet Cecal weight, g Cecal pH Acetic acid, mg/g cecum Cecal weight, g Cecal pH Acetic acid, mg/g cecum
18 Weeks after last DMH injection 0.186 ± 0.062 (4) 7.75 ± 0.02 (2) 0.24 ± 0.02 (2) 38 Weeks after last DMH injection 0.213 ± 0.048 (14) 7.19 ± 0.08 (2) 0.49 ± 0.20 (2)
Bifidogenic diet 0.411 ± 0.057* (4) 7.33 ± 0.08f (2) 0.63 ± 0.23 (2) 0.440 ± 0.116^13) 6.99 ± 0.14 (2) 0.89 ± 0.10 (2)
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a: Nos. in parentheses represent no. cif pooled observations. b: All comparisons were made within horizontal rows. Statistical significance is as follows: * ,p< 0.01; t, p < 0.05; t,P < 0.0005.
used as a dietary adjunct. Consumption of the bifidogenic diet significantly increased the logarithmic ratio of anaerobes to aerobes. This ratio has been generally suggested as an indicator for intestinal bacterial environment, with a higher ratio reflecting a healthier condition (20). The expected changes in cecal weight and pH were observed. The increase in cecal weight in animals fed the bifidogenic diet was mainly due to an increase in the amount of fluid present in the cecal content as a result of high osmotic activity of Neosugar. On the basis of previous studies (7), a 5% level of Neosugar was chosen to provide a source of selective substrate for Bifidobacteria without causing the side effect of severe cecal enlargement. Moderate cecal enlargement is considered to be a normal homeostatic response and does not associate with any pathological sequelae (21). Although cecal acetic acid concentrations were not statistically different between the two treatment groups, their total amount present in the ceca of animals fed bifidogenic diet were considerably larger because of the significantly greater cecal weight of those animals. It is likely that, besides acetic acid, other short-chain fatty acids produced by Bifidobacteria during the fermentation of Neosugar may contribute to the lower pH in the Bifidobacteria-fed animals. In view of the effective buffering action of bicarbonate ions in the colon (22), regular consumption of Bifidobacteria and bifidogenic substrate is necessary for the maintenance of an acidic intestinal environment. Consumption of the bifidogenic diet appeared to provide possible protection against chemically induced colonic carcinogenesis in CFj mice, as seen from the reduction in the number of aberrant crypts. The divergence of the slopes of aberrant crypt number in the two treatment groups suggested that the protective effect of Bifidobacteria might be progressively stronger with the carcinogenic process. Distribution of aberrant crypts and foci indicated that consumption of the bifidogenic diet might confine the aberrance in the more distal end of the large intestine. The lack of aberrant crypts presented in the cecal mucosa in animals receiving Bifidobacteria may be the result of its proximity to the cecum and the proximal colon, where the bacterial fermentation activity was strongest. In a time course study where CFj mice were killed two to four weeks after a single injection of the carcinogen azoxymethane, it was reported that the distribution of aberrant foci in the rectal section decreased from 90% in Week 2 to 71% in Week 4 while those in the midcolon section increased from 10% to 29% (15). Therefore, it would seem that the aberrant crypts start to appear at the more distal end of the colon and progress toward the cecum with time. Several hypotheses have been suggested for the protective effect of Bifidobacteria against DMH-induced colonic carcinogenesis. The most probable one is the ability of Bifidobacteria
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to lower the intraluminal pH through the production of short-chain fatty acids, in particular, acetic acid and lactic acid. Acetic acid was reported to be a more potent antagonistic agent against Gram-negative bacteria than lactic acid or hydrochloric acid. Although a pH of less than 4 with hydrochloric acid was required to inhibit the growth of Escherichia coli, acetic acid showed a similar effect at pH 5.8 (23). Thorton (24) suggested that colonic pH is an important factor in colonic carcinogenesis and that a low intraluminal pH may have a protective effect. Samelson and colleagues (25) also reported that the DMH-treated rats with acid stool, achieved by the consumption of lactulose or sodium sulfate, developed fewer tumors than the controls. Epidemiological studies also showed an association between a reduced colon cancer risk and a low fecal pH (26). In vitro studies have shown that bile acids do not undergo 7a-dehydroxylation reaction at pH below neutrality (27). Animal studies also showed that low pH may inhibit the bacterial conversion of primary bile acids into cocarcinogenic deoxycholic and lithocholic acids (4,28,29). In a study on patients with adenomas, a lower intestinal pH induced by the consumption of lactulose was found to significantly reduce deoxycholate and increase primary acid concentration (30). Low pH may also inhibit nuclear dehydrogenase activity, which may be responsible for the conversion of bile acids to the carcinogenic cyclopentaphenanthrenes (31). Furthermore the damaging effect of deoxycholic acid on perfused colon was reported to be markly higher at pH 7.9 than 5.9 (32). High acidity in the colon may stimulate intestinal peristalsis and colonic mucus production, which may reduce the contact of carcinogens and cocarcinogens with colonocytes (33,34). In conclusion, feeding of Bifidobacteria and the bifidogenic factor Neosugar appeared to mitigate the process of colonic carcinogenesis, as shown by a reduction in the number of aberrant crypts in the colon. Intestinal intraluminal acidification and displacement of undesirable bacteria could be responsible for this effect. Dietary practices such as ingestion of cultured dairy products that favor the predominance of Bifidobacteria may play an important role in the health of the intestinal environment and the prevention of chronic diseases. Acknowledgments and Notes Financial support was provided by Gelda Scientific and Industrial Development (Mississauga, Ontario, Canada) and the National Research Council of Canada (Ottawa, Ontario, Canada). Address reprint requests to Dr. A. V. Rao, Dept. of Nutritional Sciences, University of Toronto, 150 College St., Toronto, Ontario M5S 1A8, Canada. Submitted 10 January 1991; accepted in final form 19 June 1991.
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Long‐term effect of bifidobacteria and Neosugar on precursor lesions of colonic cancer in cf1 mice a
Malcolm Koo & A. Venketeshwer Rao
a
a
Department of Nutritional Sciences, Faculty of Medicine, University of Toronto, 150 College St., Toronto, Ontario, M58 1A8, Canada Version of record first published: 04 Aug 2009.
To cite this article: Malcolm Koo & A. Venketeshwer Rao (1991): Long‐term effect of bifidobacteria and Neosugar on precursor lesions of colonic cancer in cf1 mice, Nutrition and Cancer, 16:3-4, 249-257 To link to this article: http://dx.doi.org/10.1080/01635589109514163
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Long-Term Effect of Bifidobacteria and Neosugar on Precursor Lesions of Colonic Cancer in CF1 Mice Downloaded by [Universite Laval] at 22:50 19 April 2013
Malcolm Koo and A. Venketeshwer Rao
Abstract This investigation was undertaken to study the role of Bifidobacteria and bifidogenic factor Neosugar in the process of 1,2-dimethylhydrazine-induced colonic carcinogenesis in CF1 mice. Intestinal colonization and selective proliferation of Bifidobacteria were achieved by oral administration of indigenous Bifidobacteria and the incorporation of 5% Neosugar in the diet of animals. The Bifidobacteria were isolated from the feces of CF, mice and were identified to be Bifidobacterium pseudolongum biovar b. This incidence of aberrant crypts andfoci were significantly lower 38 weeks after the last injection of the carcinogen in animals fed Bifidobacteria than in animals treated with the carcinogen alone. The aberrance also appeared to be confined to the more distal end of the colon in animals fed bifidogenic diet. Such changes in the precursor lesions of colonic carcinogenesis are presumably due to the increase in the number of Bifidobacteria and their acidifying action in the lower intestinal tract of the animals. (Nutr Cancer 16, 249-257, 1991)
Introduction
Intestinal bacteria play an important role in the process of colonic carcinogenesis. Major evidence supporting this view comes from observations of lower incidence of chemically induced colon cancer in germ-free animals (1,2) and the ability of intestinal bacteria to produce various possible carcinogens and promoters (3). With respect to enhanced cancer risk, secondary bile acids (4) and cholesterol metabolites (5) received the greatest attention. Not all intestinal bacteria, however, are associated with the etiology of colon cancer. Lactobacilli have been reported as being protective against chemically induced colonic carcinogenesis in rats (6). Similarly, Bifidobacteria, which are also acid-producing bacteria, have been shown to be protective against colonic carcinogenesis (7). It was reported that feeding of naturally fermented sour milk but not artificially acidified milk in the diet significantly reduced the number of colonic tumors induced by dimethylhydrazine in rats (8). The authors are affiliated with the Department of Nutritional Sciences, Faculty of Medicine, University of Toronto, Toronto, Ontario M58 1A8, Canada.
Copyright © 1991, Lawrence Erlbaum Associates, Inc.
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In the same study, a parallel increase in fecal bifidobacterial count was also observed. In addition to the intraluminal acidification effect, possible antibacterial activity of Bifidobacteria might be responsible for the antitumorigenic effect of sour milk. Bifidobacteria are Gram-positive anaerobic bacteria. They constitute a major part of the normal human intestinal and fecal flora throughout life. They appear in the stools two to five days after birth and rapidly establish themselves to be the most predominant bacterial group in the colon, ranging from 109 to 1011 cells/g feces. However, their number decreases significantly during senescence (9,10). Bifidobacteria are unique in their metabolism of carbohydrates. They produce acetic and L-(+)-lactic acids in a molar ratio of 3:2 from glucose through the characteristic fructose 6-phosphate shunt (11). To enhance the selective proliferation and colonization of Bifidobacteria in the intestinal tract, a bifidogenic factor, Neosugar, was incorporated into the diet of animals receiving Bifidobacteria orally (12). Neosugar is a mixture of fructooligosaccharides that is not digested by gastrointestinal enzymes of humans or animals (13) but readily utilized by certain intestinal bacteria including Bifidobacteria, Bacteroides fragilis, Peptostreptococcus spp, and Klebsiella pneumoniae. Acetic, propionic, and butyric acids are produced by these bacteria, which may substantially reduce the intestinal pH (14). Neosugar, on the other hand, is not used by other undesirable bacteria such as Escherichia coli, Clostridium perfringes, and C. paraputrificum (12). The principal objective of this study was to investigate the effect of a bifidogenic regimen consisting of oral administration of viable Bifidobacteria and dietary supplementation of Neosugar on 1,2-dimethylhydrazine-induced colonic carcinogenesis in CFj mice. Aberrant crypt in the colon was used to assess the carcinogenic process. When two or more aberrant crypts are found adjacent to each other, the resulting cluster is termed aberrant foci. McLellan and Bird (17) showed that a single crypt may be replaced by multicrypt foci from two to four weeks after the final injection. They suggested that multicrypt foci may be associated with higher doses of carcinogen. However, the exact relationship between single-crypt and multicrypt foci is not clear. Both parameters were determined in the present study. Several lines of evidence indicate that aberrant crypts represent precursor lesions of chemically induced colon cancer (15). Aberrant crypts exhibit many common characteristics of precursor lesions. They are reported to be specifically induced by colon carcinogens, and the induction has been reported to be dose dependent. The frequency of aberrant crypts is modulated by agents that modify the carcinogenic process, often with the degree observed when colonic tumors are the end points. In addition, an increased proliferation rate, which is common to early changes in colon carcinogenesis, is observed in aberrant crypts. Methods Experimental Design Three experimental groups were established: mice treated with 1,2-dimethylhydrazine (DMH) and fed Bifidobacteria and a bifidogenic diet (« = 20), mice treated with DMH and fed a normal diet {n = 21), and control mice that did not receive DMH and were fed a normal diet (n = 5). The normal diet used was AIN-76A (ICN Biomedicals, Cleveland, OH), and 5% Neosugar P (Meiji Seika Kaisha, Tokyo, Japan) was added to the AIN-76A diet to form the bifidogenic diet. Female CFj mice (Charles River, Montreal, Quebec) weighing 21.4 ± 1.5 g were used in the study. They were placed on their respective diets three days after arrival. After a two-week acclimation period, treatment mice were given the first of the six weekly subcutaneous injections of DMH solution (20 mg/kg body wt; Aldrich Chemical, Milwaukee, WI). The DMH solution was freshly made each time before injection in 0.001 M
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ethylenediaminetetraacetic acid (EDTA, Sigma Chemical, St. Louis, MO) solution and brought to pH 6.5 with sodium bicarbonate. Control animals were injected likewise with EDTA solution. One week after the last injection, oral intubation of viable bifidobacterial suspension (lOVmouse) twice a week was started. Animals were killed at three time points: 18, 28, and 38 weeks after the last carcinogen injection. Ten animals, four from each treatment group and two from the control, were killed at Week 18 by cervical dislocation. Colons were fixed in 10% phosphate-buffered formalin and quantified for the presence of aberrant crypts. Ceca along with their content were analyzed for their pH and acetic acid content. Seven mice, three from each treatment group and one from the control, were killed at Week 28 for aberrant crypt determination. The remaining animals were killed 38 weeks after the last injection of the carcinogen. Overnight fecal samples were collected for the enumeration of total aerobes, total anaerobes, and Bifidobacteria. Ceca and their content were analyzed for acetic acid content, and colons were analyzed for the presence of aberrant crypt.
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Bacteriological Preparation Bifidobacterial suspension used in the oral intubation was isolated from the feces of CV1 mice. Overnight fecal sample was collected, homogenized in peptone water (GIBCO Laboratories Life Technologies, Madison, WI), and streaked on blood-base glucose blood liver agar for selective isolation of Bifidobacteria. The composition of the blood-base glucose blood liver agar is shown in Table 1. The inoculated plates were incubated in anaerobic jars at 37°C for 72 hours. The anaerobic requirement of Bifidobacteria was satisfied by the use of Anaerocult C Gas Pak (Merck, Darmstadt, Germany). After incubation, all distinct types of colonies were examined under a microscope and subjected to further biochemical analysis and the fructose 6-phosphate phosphoketolase enzymatic confirmation of Bifidobacteria (16). Identification of Bifidobacteria at the species level was achieved by matching the carbohydrate utilization Table 1. Composition of Glucose Blood Liver Agar" Ingredient Meat extract Proteose peptone Peptone Soy bean peptone Yeast extract Liver extract powder Glucose Soluble starch K 2 HPO 4 KH2PO4 MgSO4-7H2O NaCl MnSO4 Defoaming agent Polysorbate 80 L-Cysteine hydrochloride Agar Blood agar base
Composition* 2.4 10.0 3.0 3.0 5.0 3.2 10.0 0.5 1.0 1.0 0.01 0.01 0.007 0.2 1.0 0.3 15.0 40.0
a: Formula based on Mitsuoka (35) with slight modifications. b: Expressed in grams per liter of water.
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profile of isolated Bifidobacteria with that in the literature (16) by means of the API system (API Laboratory Products, St. Laurent, Quebec). Stock culture of the isolated Bifidobacteria was maintained on Lactobacillus MRS agar. The agar was prepared by adding 55 g of Lactobacillus MRS broth (Difco Laboratories, Detroit, MI) and 15 g of agar (GIBCO Laboratories Life Technologies) to 1 liter of distilled water. Cysteine hydrochloride (Sigma Chemical) was added to the medium after it was autoclaved to a final concentration of 0.03%. The cell suspension used in oral intubation was made by suspending saline-washed 48-hour culture broth in phosphate-buffered saline (pH 6.5) to a concentration of at least 109 viable cells per milliliter. Bacterial Enumeration
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Samples were homogenated and serially diluted with peptone water for plating in a standard fashion. Plate count agar (Difco Laboratories) was used for enumeration of total aerobic bacteria, and reinforced clostridial agar (Difco Laboratories) was used for total anaerobic bacterial count. Bifidobacteria were enumerated in Lactobacillus MRS agar. All plates were incubated at 37°C for 48 hours in appropriate atmospheric conditions. Laboratory Procedures Cecal samples were homogenized using a vortex mixer with nine volumes of physiological saline in capped polypropylene centrifuge tubes. The air in the tubes was replaced by nitrogen. The homogenates were then centrifuged at 3,000 rpm (=1,650 g) for 15 minutes. The pH of the supernatants was measured immediately. Acetic acid concentration of the cecal samples was quantified by the enzymatic ultraviolet method (Boehringer Mannheim Canada, Dorval, Quebec). Aberrant crypts were scored using the procedure described by McLellan and Bird (17). Colonic samples were stained with methylene blue dye (1% in phosphate-buffered saline) for 10 minutes. The mucosal surfaces were examined under X100 magnification with a light microscope. To determine the distribution of aberrant crypts, colonic mucosa 5 cm anterior to the anus was divided into three sections where R represented the first 2 cm from the rectal end, M was the next 2 cm, and C was the 1st cm from M. Criteria used for identification of aberrant crypts were elongation and increased size of crypt cells, thickening of epithelial lining, and increased pericryptal zone (18). Statistical Methods All results were subjected to Student's / test by means of the SAS statistical package (SAS Institute, Cary, NC). The Satterthwaite approximation was used for samples with unequal variances. All results were expressed as means ± SD. Statistical comparisons of bacterial counts were performed on logarithmic-transformed raw data, and the results were expressed as logarithm to the base 10 of the number of viable bacteria per gram of feces. Results No aberrant crypts were identified in the colons of control animals at all three times of termination. One adenocarcinoma was found in one of the mice in the carcinogen-treated group 28 weeks after the last carcinogen injection; otherwise, all animals were in good general condition. Deschner and Long (19), using identical carcinogen, animals, and injection protocol, reported that 83% of the animals were tumor bearing, with a frequency of 2.1 macroscopically visible colonic tumors per mouse between the 20th and 45th week of the experiment. The extremely low tumor incidence observed in the present study is somewhat surprising. A higher tumor incidence may be seen if the experiment had continued
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for another two to three months. DMH was the choice of carcinogen because it induces lesions mainly in the distal colon and follows the adenomatous polyps-adenocarcinoma sequence similar to that observed in humans. The number of injections was kept to a minimum to avoid the carpeting phenomenon of colonic tumors, which may otherwise overwhelm any protective effect contributed by Bifidobacteria. Taken together, the mean numbers of aberrant crypts and aberrant foci were lower in animals receiving the bifidogenic diet. The incidence of occurrence was 100% in both treatment groups. Initially, at Week 18, the mean number of aberrant crypts was significantly lower in animals fed the bifidogenic diet. At Week 18 the mean number of aberrant foci was statistically lower in animals fed the bifidogenic diet, and at Week 38 both parameters were significantly lower in these animals. The trends of the mean aberrant crypts and foci are shown in Figures 1 and 2, respectively. There appears to be a diverging trend between the two groups of animals in terms of the incidence of both aberrant crypts and foci. The distribution of aberrant foci is shown in Figure 3. A similar pattern of distribution was also observed in aberrant crypts. The majority of the aberrant foci were found at the mucosa of the midcolon. Starting from Week 28, aberrant foci began to appear in the cecal mucosa exclusively in the mice treated with DMH alone. At Week 38, the mean bifidobacterial count in the feces of animals fed bifidogenic diet was increased from 8.85 ± 0.20 to 9.45 ± 0.19 (p < 0.05). The logarithmic ratio of fecal anaerobes to aerobes was also elevated from 0.20 ± 0.01 to 0.92 ± 0.34 (p < 0.05). Mean cecal weight, pH, and acetic acid content of the animals are summarized in Table 2. Mean full cecal weights of animals fed bifidogenic diet were significantly (p < 0.05) higher in Weeks 18 and 38. Cecal pH was significantly lower in these animals (p < 0.001) at Week 18, but the reduction was not significant at Week 38. Although cecal acetic acid concentration in bifidogenic diet-fed animals was almost twice that of the other group, the changes were not statistically significant.
40 35 C
30
O O
25
>
20
6 -
15 10
Figure 1. Effect of bifidogenic diet on nos. of aberrant crypts in 1,2-dimethylhydrazine (DMH)-treated CF, mice. Horizontal axis, weeks after last injection of DMH. Symbols are as follows: a, p < 0.05; b,/> < 0.01.
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10
15 20 25 30 35 40 Time (week)
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10
15
20
35 40
25
Time (week) 100%
Figure 2. Effect of bifidogenic diet on nos. of aberrant foci in 1,2-dimethylhydrazine (DMH)-treated CF, mice. Horizontal axis, weeks after last injection of DMH. Symbols are as follows: a, p < 0.05; b , p < 0.01.
n n
Distribution of Aberrant Foci
75%-
50%-
25% - !
Bifido
DMH
Bifido
Week 18 t
) Rectal mucosa
• •
Cecal mucosa
DMH
Week 28 I
Bifido
DMH
Week 3 8
.1 Midcolon mucosa
Figure 3. Effect of bifidogenic diet on distribution of aberrant foci in 1,2-dimethylhydrazine (DMH)-treated CF, mice. Horizontal axis, weeks after last injection of DHM in animals fed bifidogenic and normal diet. Aberrance was confined to more distal end of colon in mice fed bifidogenic diet at all 3 time points.
Discussion
Feeding of indigenous Bifidobacteria was effective in significantly increasing their number in the lower intestinal tract of the animals. Indigenous strains of Bifidobacteria present in the feces of the animals are presumably those that have already possessed or acquired desirable characteristics for a successful colonization in the intestine of their host. The matching of host specificity may therefore facilitate a rapid proliferation of Bifidobacteria when they are
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Table 2. Effect of Bifidogenic Diet on Full Cecal Weight, Cecal. pH , and Acetic Acid in DMH-Treated CFj Mice"'" DMH Treated Normal diet Cecal weight, g Cecal pH Acetic acid, mg/g cecum Cecal weight, g Cecal pH Acetic acid, mg/g cecum
18 Weeks after last DMH injection 0.186 ± 0.062 (4) 7.75 ± 0.02 (2) 0.24 ± 0.02 (2) 38 Weeks after last DMH injection 0.213 ± 0.048 (14) 7.19 ± 0.08 (2) 0.49 ± 0.20 (2)
Bifidogenic diet 0.411 ± 0.057* (4) 7.33 ± 0.08f (2) 0.63 ± 0.23 (2) 0.440 ± 0.116^13) 6.99 ± 0.14 (2) 0.89 ± 0.10 (2)
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a: Nos. in parentheses represent no. cif pooled observations. b: All comparisons were made within horizontal rows. Statistical significance is as follows: * ,p< 0.01; t, p < 0.05; t,P < 0.0005.
used as a dietary adjunct. Consumption of the bifidogenic diet significantly increased the logarithmic ratio of anaerobes to aerobes. This ratio has been generally suggested as an indicator for intestinal bacterial environment, with a higher ratio reflecting a healthier condition (20). The expected changes in cecal weight and pH were observed. The increase in cecal weight in animals fed the bifidogenic diet was mainly due to an increase in the amount of fluid present in the cecal content as a result of high osmotic activity of Neosugar. On the basis of previous studies (7), a 5% level of Neosugar was chosen to provide a source of selective substrate for Bifidobacteria without causing the side effect of severe cecal enlargement. Moderate cecal enlargement is considered to be a normal homeostatic response and does not associate with any pathological sequelae (21). Although cecal acetic acid concentrations were not statistically different between the two treatment groups, their total amount present in the ceca of animals fed bifidogenic diet were considerably larger because of the significantly greater cecal weight of those animals. It is likely that, besides acetic acid, other short-chain fatty acids produced by Bifidobacteria during the fermentation of Neosugar may contribute to the lower pH in the Bifidobacteria-fed animals. In view of the effective buffering action of bicarbonate ions in the colon (22), regular consumption of Bifidobacteria and bifidogenic substrate is necessary for the maintenance of an acidic intestinal environment. Consumption of the bifidogenic diet appeared to provide possible protection against chemically induced colonic carcinogenesis in CFj mice, as seen from the reduction in the number of aberrant crypts. The divergence of the slopes of aberrant crypt number in the two treatment groups suggested that the protective effect of Bifidobacteria might be progressively stronger with the carcinogenic process. Distribution of aberrant crypts and foci indicated that consumption of the bifidogenic diet might confine the aberrance in the more distal end of the large intestine. The lack of aberrant crypts presented in the cecal mucosa in animals receiving Bifidobacteria may be the result of its proximity to the cecum and the proximal colon, where the bacterial fermentation activity was strongest. In a time course study where CFj mice were killed two to four weeks after a single injection of the carcinogen azoxymethane, it was reported that the distribution of aberrant foci in the rectal section decreased from 90% in Week 2 to 71% in Week 4 while those in the midcolon section increased from 10% to 29% (15). Therefore, it would seem that the aberrant crypts start to appear at the more distal end of the colon and progress toward the cecum with time. Several hypotheses have been suggested for the protective effect of Bifidobacteria against DMH-induced colonic carcinogenesis. The most probable one is the ability of Bifidobacteria
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to lower the intraluminal pH through the production of short-chain fatty acids, in particular, acetic acid and lactic acid. Acetic acid was reported to be a more potent antagonistic agent against Gram-negative bacteria than lactic acid or hydrochloric acid. Although a pH of less than 4 with hydrochloric acid was required to inhibit the growth of Escherichia coli, acetic acid showed a similar effect at pH 5.8 (23). Thorton (24) suggested that colonic pH is an important factor in colonic carcinogenesis and that a low intraluminal pH may have a protective effect. Samelson and colleagues (25) also reported that the DMH-treated rats with acid stool, achieved by the consumption of lactulose or sodium sulfate, developed fewer tumors than the controls. Epidemiological studies also showed an association between a reduced colon cancer risk and a low fecal pH (26). In vitro studies have shown that bile acids do not undergo 7a-dehydroxylation reaction at pH below neutrality (27). Animal studies also showed that low pH may inhibit the bacterial conversion of primary bile acids into cocarcinogenic deoxycholic and lithocholic acids (4,28,29). In a study on patients with adenomas, a lower intestinal pH induced by the consumption of lactulose was found to significantly reduce deoxycholate and increase primary acid concentration (30). Low pH may also inhibit nuclear dehydrogenase activity, which may be responsible for the conversion of bile acids to the carcinogenic cyclopentaphenanthrenes (31). Furthermore the damaging effect of deoxycholic acid on perfused colon was reported to be markly higher at pH 7.9 than 5.9 (32). High acidity in the colon may stimulate intestinal peristalsis and colonic mucus production, which may reduce the contact of carcinogens and cocarcinogens with colonocytes (33,34). In conclusion, feeding of Bifidobacteria and the bifidogenic factor Neosugar appeared to mitigate the process of colonic carcinogenesis, as shown by a reduction in the number of aberrant crypts in the colon. Intestinal intraluminal acidification and displacement of undesirable bacteria could be responsible for this effect. Dietary practices such as ingestion of cultured dairy products that favor the predominance of Bifidobacteria may play an important role in the health of the intestinal environment and the prevention of chronic diseases. Acknowledgments and Notes Financial support was provided by Gelda Scientific and Industrial Development (Mississauga, Ontario, Canada) and the National Research Council of Canada (Ottawa, Ontario, Canada). Address reprint requests to Dr. A. V. Rao, Dept. of Nutritional Sciences, University of Toronto, 150 College St., Toronto, Ontario M5S 1A8, Canada. Submitted 10 January 1991; accepted in final form 19 June 1991.
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