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Nutrition and Carcinogenesis Laboratory, Department of Food and Animal Sciences, Alabama A&M University, Normal, AL 35762
2To whom correspondence should be addressed. mverghese{at}aamu.edu.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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KEY WORDS: • aberrant crypt foci • azoxymethane • colon carcinogenesis • inulin • mature rats
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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). Human metabolic and laboratory animal model studies indicate that the composition and physical properties of dietary fiber affect its beneficial effects in relation to colon cancer development (2
). Alterations in colonic cell proliferation have been observed with changes in type and amount of dietary fiber (3
). It has been suggested (4
) that the mechanisms by which dietary fiber modulates human colon cancer incidence may involve multiple actions in the lumen and target tissue. Based on current knowledge of the pathogenesis of colon cancer, it is reasonable to conclude that consumption of a fiber-rich diet is associated with reduced risk of colon cancer. However, no definitive statements can be made about the mode of action of specific types or amounts of dietary fiber on the reduction of colon cancer (4
). Inulin and oligofructose, which are present in common foods such as chicory, onion, leek, garlic, artichoke and asparagus, are ß(2
1) D-fructans (5
,6
). They are fermented by colonic microflora and behave as soluble fibers (7
) and selectively stimulate the growth of bifidobacteria at the expense of Bacteroides, clostridia or coliforms (7
–9
). These nondigestible oligosaccharides enhance the growth of bifidobacteria and could inhibit colon carcinogenesis. Bacterial fermentation of these prebiotics in the colon produces short-chain fatty acids (SCFA),3
such as butyric acid (7
,10
), which have been shown to increase apoptosis in human colonic cell lines (11
). It has been demonstrated recently that inulin also increases the apoptotic index in the colon (12
,13
). Perrin et al. (14
) studied the role of butyrate-producing fibers in relation to susceptibility to colon cancer and concluded that a colonic ecosystem related to selected fibers producing large amounts of butyrate may have a trophic effect in the large intestine and therefore be less conducive to colon carcinogenesis. Reddy and Rivenson (15
) have reported alterations in colonic microflora, which have an inhibitory effect on tumor incidence and/or growth.
Preneoplastic aberrant crypt foci (ACF) induced by azoxymethane (AOM) have been used extensively to investigate nutritional modulation of colon carcinogenesis in rats (16
). AOM is a metabolite of the procarcinogen 1,2-dimethylhydrazine and is one metabolic step closer to the proximate carcinogen capable of inducing colonic ACF. The weanling rat is the most commonly used model. Because cancer is generally a disease of aged populations, mature rat models may be more appropriate. However, very little published information is available on the use of mature rats in studying ACF. Paulsen et al. (17
) reported more ACF in the colon of adult rats than in pups who received two subcutaneous injections of AOM at 3.75 mg/kg body.
This study was designed to determine the effects of feeding dietary inulin at different levels (0, 2.5, 5.0 and 10 g/100 g) on AOM-induced colonic ACF in mature Fisher 344 male rats.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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Mature (12 mo) Fisher 344 retired male breeder rats obtained from the National Institute on Aging, Bethesda, MD were studied. In experiment 1, groups of rats were assigned to six doses of AOM (0, 4, 8, 10, 12 and 16 mg/kg body) and fed AIN-93M diet (18
) for 11 wk after a 2-wk acclimation period. In experiment 2, four groups of 12 rats each were assigned to the following: AIN-93M (Control-C) and AIN-93M with 2.5% inulin, AIN-93M with 5% inulin and AIN-93M with 10% inulin added at the expense of cornstarch. The diets were fed for 11 wk after a 2-wk acclimation period. The composition of the AIN-93M diet is shown in Table 1
. Long-chain inulin extracted from chicory roots was obtained from ORAFTI (Tienen, Belgium) as Raftiline®, with a degree of polymerization of 10–65 with an average chain length of 25. Temperature and relative humidity were maintained at 21 ± 1°C and 50%, respectively. Light and dark cycles were 12 h each. Rats consumed feed and water ad libitum. Weekly body weights and daily feed intakes were recorded. The diets were prepared fresh each week and stored at 4°C until fed. Diets were formulated on the basis of AIN-93M diets, and the ingredients were obtained from ICN (Costa Mesa, CA). The Institutional Animal Care and Use Committee of Alabama A&M University approved all the protocols involving rats.
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All rats were fed two subcutaneous injections of AOM in saline (Sigma-Aldrich, St. Louis, MO), one at 55 wk and another at 56 wk of age. On the basis of the mortality rate and the rate of ACF induction in experiment 1, the dose of AOM used in experiment 2 was 10 mg/kg body weight. Rats were killed by CO2 asphyxiation at the end of an 11-wk feeding period.
Diarrheal index.
Diarrheal index was measured by assigning the following numbers based on the appearance of the pellets: 0, normal; 1, mild diarrhea (soft pellets); 2, moderate diarrhea (semisolid pellets); 3, overt diarrhea (pasty pellets); and 4, severe diarrhea (watery feces).
Cecal weight and pH.
The cecum from each rat was excised, weighed and split open, and the pH of the contents was recorded.
Counting ACF.
The colon of each rat was removed and flushed with potassium phosphate buffer (0.1 mol/L, pH 7.2) and scored (19
) for ACF as described by Bird (20
).
Statistical analysis.
Values are given as mean ± SEM. Data were analyzed using the SAS statistical program by analysis of variance and means were separated using the Tukey’s studentized range test (21
). Differences were considered significant at P < 0.05, unless otherwise indicated. Differences between control and inulin fed groups and proximal and distal colon were tested by Student’s t test and paired t test, respectively. Regression analysis was also conducted.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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Survival rates for rats administered 4, 8 and 10 mg of AOM/kg body were not different from that of the control. However, the survival rates in rats given the 12 and 16 mg doses/kg were lower (P < 0.05), with rats given the 16 mg dose/kg having only a 25% survival rate (Table 2)
. Body weight gains were reduced (P < 0.05) in rats administered 4–12 mg of AOM/kg. Feed intake ranged from 14.8 to 15.5 g/d and did not differ among groups.
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). Crypt multiplicity as measured by crypts per aberrant crypt focus is shown in Figure 1
. The groups administered AOM at 4 and 8 mg/kg body did not differ in the number of foci with 1, 2 and 
5 crypts. The number of foci with 2 and 5 crypts were higher (P < 0.05) in the group given AOM at 16 mg/kg body than in all other groups. AOM doses of 10, 12 and 16 mg/kg body induced more foci with 3, 4 and 5 crypts than the 4 and 8 mg/kg doses, indicating a shift to larger ACF at higher doses (Fig. 1)
. On the basis of the mortality rate and the rate of ACF formation, a dose of 10 mg/kg body was selected for use in experiment 2.
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Body weight and feed intake.
Body weight gains of rats were lower (P < 0.05) in the 10 g/100 g inulin group compared with the controls (Table 3)
. The initial weight of the mature rats was 403.0 ± 4.3 g. Daily feed intakes did not differ among the groups and ranged from 14.8 ± 2.10 to 15.9 ± 1.67 g/d. However, feed intake was lower than previously observed in weanling rats (19
).
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0.001) compared with the control group (Table 3)
. Cecal pH was dramatically lowered in rats fed the 5 and 10% inulin diets compared with those fed the control diet (P 0.001). There was a positive correlation (R2 = 0.9135), (P < 0.05) between dietary inulin level and cecal weight, and a negative correlation (R2 = 0.9152), (P < 0.05) between dietary inulin and cecal pH. Stool consistency was firm (pelleted) throughout the study, with no visible difference among groups.
Aberrant crypt foci.
In rats fed the control diet, AOM induced 162.3 ± 1.72 ACF/colon. The percentage of reductions of ACF in the rats fed 2.5, 5 and 10 g/100 g inulin diets were 25, 51 and 65, respectively (P < 0.05). There were 24, 52 and 69% reductions in total crypts (Table 4)
in the rats fed inulin at 2.5, 5 and 10 g/100 g, respectively. As expected, ACF in the distal colon of all groups were more numerous than in the proximal colon. These data are consistent with reports that the distal colon has a greater colon cancer incidence than the proximal colon in humans (22
). There were linear relationships (P < 0.05) between the dose of inulin in the diet and the number of ACF and total crypts in the colon (Fig. 2
). The total number of foci containing 1, 2, 3, 4 and 5 crypts per focus were counted in the proximal and distal regions of the colon (Fig. 3
). The foci with 3, 4 and 5 crypts were lower (P < 0.05) in the rats fed the 5 and 10 g/100 g inulin diets than in the control group. The number of foci with 2 crypts did not differ among the inulin fed groups.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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). Also, mature rats appeared to be more sensitive to AOM with respect to ACF formation than younger rats (19
). Verghese (23
) reported a mean of 150 ACF in the colon of younger rats fed an AOM dose of 16 mg/kg body, whereas in this study the number of ACF in the colon was 160 at an AOM dose of 10 mg/kg body. Paulsen et al. (17
) also reported more ACF in the colon of adult rats than in pups receiving two subcutaneous injections of AOM at 3.75 mg/kg body.
The suitability of mature rats as a model for studies of nutritional modulation of carcinogenesis was tested by using long-chain dietary inulin, a suppressor of AOM-induced ACF in younger rats (23
,24
).
In the present study, feeding inulin at 2.5, 5 and 10 g/100 g suppressed total colonic ACF as well as multi-crypt foci formation in mature rats. Inulin and oligofructose are natural food ingredients present in many edible plants, such as onion, garlic, asparagus, wheat, leek, chicory, artichoke and banana (3
,25
). The results of a previous experiment (23
) suggested that 5 and 10 g/100 g inulin reduced ACF and colon tumors in weanling rats. In the present study, there was a clear dose response in suppression of ACF and total crypt formation with increasing levels of dietary inulin (Fig. 2)
. The trends in the reduction of ACF at different inulin levels (2.5, 5 and 10 g/100 g) were similar when comparisons were made among treatments (Table 4)
in the same region (proximal or distal colon). The number of crypts/100 g body for rats fed the control, 2.5, 5 and 10 g/100 g inulin diets were 37.5, 28.3, 18.0 and 13.3, respectively. These data indicate that the reduced weight gain in the 10 g/100 g inulin group was not responsible for the lesser number of ACF in this group because the 5 g/100 g inulin group had the greatest weight gain and showed similar reductions. Unlike in younger rats (23
), there was no diarrhea in mature rats. This probably reflects the fact that mature rats have diverse gastrointestinal microflora and handle inulin better than weanling rats.
This is the first study providing evidence that ingestion of inulin inhibits colonic ACF in mature rats. The precise mechanisms by which inulin inhibits preneoplastic lesions of the colon are not fully known. Gibson and Roberfroid (7
) and Gibson et al. (8
) have suggested that the effect of inulin proceeds through the modulation of microflora and production of SCFA in the colon. Challa et al. (26
) have shown the inhibitory effects of bifidobacteria on AOM-induced ACF. In addition to being bifidogenic, Campbell et al. (10
) reported that inulin increases the production of SCFA (10
,27
), especially butyrate, in the colon by microbial fermentation (7
). Hughes and Rowland (12
) observed a significant increase in the colonic apoptotic index in rats fed 5% inulin. They also suggested that butyrate is central in stimulating this anticarcinogenic property. Feeding of fructo-oligosaccharides produces large amounts of butyric acid, which seems to have a protective role in relation to susceptibility to colon cancer (14
). High butyrate levels following fermentation of soluble fibers may inhibit events in colon tumorigenesis by controlling the transcription expression and activity of key proteins involved in the apoptotic cascade (28
). McIntosh (29
) reported significantly fewer adenocarcinomas in rats with higher cecal butyrate concentrations, suggesting a possible protective influence of butyrate. In addition, cecal ß-glucuronidase activity was also inversely proportional to the number of adenomas in the colon, suggesting the enhanced protection by a higher ß-glucuronidase activity. Although butyrate may have antineoplastic activities, it can not be the sole reason for the protective effect of a fiber because distal colonic butyrate concentrations alone are not protective of colon cancer development (30
).
Rowland et al. (31
) did not observe any changes in cecal pH or cecal weight in rats fed Bifidobacterium longum without inulin, although they reported a significant increase in cecal weight (20–32%) and a significant decrease in pH in those fed 5% inulin. Similar results were demonstrated in adult rats in this study suggesting that consumption of inulin is associated with potentially beneficial changes in cecal physiology and bacterial metabolic activity thus lowering the incidence of putative preneoplastic lesions and tumor risk in the colon.
In conclusion, mature rats may be used as models in studies of the nutritional modulation of AOM-induced carcinogenesis. Feeding inulin significantly reduced the total number of ACF, preneoplastic markers of malignant potential in the process of colon carcinogenesis in mature Fisher 344 male rats. The observations made in this study are supported by the results of studies with young rats (19
,31
–33
).
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FOOTNOTES |
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3 Abbreviations used: ACF, aberrant crypt foci; AOM, azoxymethane; SCFA, short-chain fatty acids.
Manuscript received 24 May 2001. Initial review completed 22 July 2001. Revision accepted 30 May 2002.
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LITERATURE CITED |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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1. Parker, S. L., Tong, T., Bolden, S. & Wingo, P. A. (1997) Cancer statistics. CA Cancer J. Clin. 47:5-27.[Medline]
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15. Reddy, B. S. & Rivenson, A. (1993) Inhibitory effect of Bifidobacterium longum on colon, mammary and liver carcinogenesis induced by 2-amino-3-methylimidazo(4,5-f)quinoline, a food mutagen. Cancer Res. 53:3914-3918.
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17. Paulsen, J. E., Fulland, R. C. & Alexander, J. (2000) Age-dependent induction of aberrant crypt foci in rat colon by 2-amino-1-methyl-6-phenylimidazo [4,5-b] pyridine and azoxymethane. Pharmacol. Toxicol. 87:69-73.[Medline]
18. Reeves, P. G., Nielsen, F. H. & Fahey, G. C., Jr. (1993) AIN-93 purified diet for laboratory rodents: final report of the American Institute of Nutrition adhoc writing committee on the reformulation of the AIN-76A rodent diet. J. Nutr. 123:1939-1951.
19. Verghese, M., Rao, D. R., Chawan, C. B. & Shackelford, L. (2002) Dietary inulin suppresses azoxymethane-induced aberrant crypt foci and colon tumors at the promotion stage in young Fisher 344 rats. J. Nutr. 132:2809-2813.
20. Bird, R. P. (1987) Observation and quantification of aberrant crypt foci in murine colon treated with a colon carcinogen: preliminary findings. Cancer Lett. 37:147-151.[Medline]
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24. Reddy, B. S. (1999) Possible mechanisms by which pro- and prebiotics influence colon carcinogenesis and tumor growth. J. Nutr. 129:1478S-1482S.
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26. Challa, A., Rao, D. R., Chawan, C. B. & Shackelford, L. (1997) Bifidobacterium longum and lactulose suppress azoxymethane-induced colonic aberrant crypt foci in rats. Carcinogenesis 18:517-521.
27. Wang, X. & Gibson, G. R. (1993) Effect of the in vitro fermentation of oligofructose and inulin by bacteria growing in the human large intestine. J. Appl. Bacteriol. 75:373-380.[Medline]
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29. McIntosh, G. H., Royle, P. J. & Pointing, G. (2001) Wheat aleurone flour increases cecal ß-glucuronidase activity an butyrate concentrations and reduces colon adenoma burden in AOM-treated rats. J. Nutr. 131:127-131.
30. Zoran, D. L., Turner, N. D., Taddeo, S. S., Chapkin, R. S. & Lupton, J. R. (1997) Wheat bran reduces tumor incidence in a rat model of colon cancer independent of effects on distal luminal butyrate concentrations. J. Nutr. 127:2217-2225.
31. Rowland, I. R., Rumney, C. J., Coutts, J. T. & Lievense, L. C. (1998) Effect of Bifidobacterium longum and inulin on gut bacterial metabolism and carcinogen-induced aberrant crypt foci in rats. Carcinogenesis 19:281-285.
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