- Email: [email protected]
Inhibitory effects of nondigestible carbohydrates of different chain lengths on azoxymethane-induced aberrant crypt foci in Fisher 344 rats
Nutrition Research 25 (2005) 859 – 868 www.elsevier.com/locate/nutres
Inhibitory effects of nondigestible carbohydrates of different chain lengths on azoxymethane-induced aberrant crypt foci in Fisher 344 rats Martha VergheseT, Lloyd T. Walker, Louis Shackelford, Chandramohan B. Chawan Nutrition and Carcinogenesis Laboratory, Department of Food and Animal Sciences, Alabama A&M University, P.O. Box 1628, Normal, AL 35762, USA Received 12 September 2004; revised 3 August 2005; accepted 1 September 2005
Abstract Colon cancer is one of the leading causes of cancer morbidity and mortality in Western countries. The objective of this study was to elucidate the effect of prebiotic carbohydrates of different chain lengths on azoxymethane-induced aberrant crypt foci in Fisher 344 male rats. After an acclimatization period of 1 week, 70 male weanling rats were divided into 7 groups and fed AIN-93G (Control) and 6 experimental diets that contained control + (maltodextrin; Raftiline HP, Raftiline ST, Raftilose P95, Raftilose Synergy1, and Mix; ORAFTI, Tienen, Belgium). All the rats received 16 mg/kg body weight of azoxymethane dissolved in saline subcutaneous at 7 and 8 weeks of age. The rats continued to receive the assigned diets until killed by carbon dioxide asphyxiation at 17 weeks of age. There was a significant ( P b .05) increase in cecal weight and a decrease in cecal pH in rats fed prebiotic carbohydrates. The highest reduction of colonic aberrant crypt foci, both in total number as well as crypt, multiplicity was seen in the group fed Mix (63.9%). Consumption of diets containing Raftilose Synergy1, Raftiline ST, and Raftiline HP showed a reduction of total colonic crypts by 52.2%, 29.6%, and 46.3%, respectively, as compared with the control diet. D 2005 Elsevier Inc. All rights reserved. Keywords: Rat; ACFs, aberrant crypt foci; AOM, azoxymethane; Fructo-oligosaccharides; SCFAs, short-chain fatty acids
T Corresponding author. E-mail address: [email protected] (M. Verghese). 0271-5317/$ – see front matter D 2005 Elsevier Inc. All rights reserved. doi:10.1016/j.nutres.2005.09.007
860
M. Verghese et al. / Nutrition Research 25 (2005) 859 – 868
1. Introduction Colon cancer is the second leading cause of cancer-related deaths. Although cancer of the colon is widely believed to be more prevalent in men, it is actually more common in women [1]. Sometimes referred to as colorectal cancer (cancer of the colon and rectum), it is predicted to be responsible for about 56 000 deaths during the year 1999. Several factors are believed to be involved in the generation of cancer. According to the American Institute for Cancer Research, some of the most common causes of cancer include heredity, tobacco use, and high consumption of alcohol. The link between diet, nutrition, and the risk for cancer is well established [2]. Studies have shown that about 50% of all cancers are the result of poor diet [3]. Diets rich in fat and poor in fiber can increase the incidence of various forms of cancer. For this reason, nutritionists recommend diets rich in largely unprocessed fruits and vegetables, which are generally low in fat and represent the source of naturally occurring dietary fiber and cancerfighting nutrients such as vitamin A, vitamin C, and carotenoids. These substances are very effective against cancer of the lung, breast, oral cavity, esophagus, stomach, pancreas, cervix, and prostate, especially the colon and rectum [4-6]. Recent research has revealed that certain classes of nondigestible carbohydrates such as inulin, which is extracted from chicory root by processes that are similar to beet sugar processing, have been shown to be chemopreventive in various experimental models [7-13]. These carbohydrates that are not digested or absorbed in the stomach or small intestine arrive almost quantitatively in the colon where they are fermented selectively by certain groups of bacteria (bifidobacteria, lactobacilli, and others). This increased bacterial metabolic activity is thought to be the basis of the anticancer properties of these prebiotic carbohydrates. The effects may be realized through increased production of short-chain fatty acids (SCFAs) through bacterial cell wall interactions with the host (immunologic) and/or reduced production of genotoxic and putrefactive compounds in the intestinal lumen. Fructooligosaccharides have been shown to be indigestible by enzymes in the human small intestine but are fermented extensively by large-bowel microflora [14,15]. There is preliminary evidence of the inhibitory effects of inulin in experimental animals [8,12,13,16]. A number of natural and synthetic compounds are being studied as potentially chemopreventive for colon cancer using the induction of aberrant crypt foci (ACFs) as the primary end point. Aberrant crypt foci are preneoplastic lesions in rat colon, and they are induced by all colon carcinogens [17,18]. Aberrant crypt foci are preneoplastic lesions found in most colon cancers [19]. Aberrant crypt foci are characterized by 1 or more crypts that appear as a single focus but are larger than normal crypts, have thickened epithelia, possess altered luminal openings, have an increased pericryptal area between them and normal crypts, and appear elevated compared with normal crypts when viewed under a microscope [20,21]. Each ACF contains numerous crypts per focus that multiply with time after treatment with a carcinogen and can be observed as early as 2 weeks after injection of the carcinogen [22]. These ACF can develop into polyps and eventually into colon cancer. Foci of aberrant crypts in rat colon are putative cancerous lesions that have been proposed as biomarkers for short-term assays for testing chemical carcinogens and chemopreventive
M. Verghese et al. / Nutrition Research 25 (2005) 859 – 868
861
agents. Here, the ability of test compounds (maltodextrin; Raftiline HP, Raftiline ST, Raftilose, Raftilose Synergy1, and Mix; ORAFTI Tienen, Belgium) to reduce the development of azoxymethane (AOM)–induced foci in the colon of male Fisher 344 rats was evaluated as a screening assay for these potential chemopreventive agents. This study was designed to determine whether (1) dietary inulin preparations of different chain lengths can reduce AOM-induced ACF in the rat colon and (2) the chemopreventive effect can be increased by combining inulin fractions (Raftiline HP and oligofructose). 2. Methods and materials 2.1. Animals, housing, and diets After 1 week of acclimatization, male Fisher 344 weanling rats were divided into 8 groups of 10 rats each and assigned the AIN-93G (Control-C) and experimental diets for 13 weeks in ACF study. Temperature and relative humidity were maintained at 218C F 18C and 50%, respectively. Light and dark cycles were for 12 hours each. Feed and water were provided ad libitum. Body weights and feed intakes were recorded weekly. The diets were prepared fresh every week and stored at 48C. Diets (Table 1) were formulated based on AIN-93G diets [23], and the ingredients were obtained from ICN Biochemicals, Costa Mesa, Calif. All the protocols involving rats have been approved by the Institutional Animal Care and Use Committee of Alabama A&M University. Prebiotic carbohydrates were obtained from ORAFTI. All other chemicals were obtained from Sigma Chemical Co (St Louis, Mo). 2.2. Prebiotic carbohydrates Various fractions of chicory inulin were used in this study. 1. Raftiline ST or native chicory inulin is a polydisperse mixture of linear molecules, all with the same basic chemical structure, which is symbolized as G-Fn Table 1 Composition of the diets fed to rats Ingredients (g/kg)
Control
Prebiotic carbohydratesa
Corn starch Prebiotic carbohydratesa Casein (85% protein) Dextrin Sucrose Fiberb Oil (soybean, no additives) Mineral mix (AIN-93G MX) Vitamin mix (AIN-93 VX) Cystine Choline bitartrate (41.1% choline)
397.5 0 200 132 100 50 70 35 10 3 2.5
297.5 100 200 132 100 50 70 35 10 3 2.5
MX = mineral mix; VX = vitamin mix. a Prebiotic carbohydrates: maltodextrin, Raftiline HP, Raftiline ST, Raftilose Synergy 1, and Mix. b Solk-FlocR, 200 FCC (FS&D, St Louis, Mo) or equivalent.
862
2. 3.
4. 5.
M. Verghese et al. / Nutrition Research 25 (2005) 859 – 868
(G indicates glucosyl moiety; F, fructosyl moiety; and n, number of fructose units linked together through b(2-1) bonds). The degree of polymerization (DP) of native chicory inulin ranges between 3 and 65, with an average degree of polymerization (DPav) of 10. Raftiline HP is inulin from which the lower DP fraction has been removed, resulting in a DPav of about 25 (also called HP-inulin). Raftilose P95 is a short-chain fraction (also called oligofructose) that is obtained by partial enzymatic hydrolysis of native chicory inulin. It is composed of linear G-Fn and F-Fn chains, with DP ranging from 2 to 8 (DPav is about 4). Raftilose Synergy1 is a 1:1 (weight per weight) co-spray-dried mixture of oligofructose and Raftiline HP. Mix is an experimental product that is composed of 1:2 (weight per weight) oligofructose-Raftiline HP.
2.3. Carcinogen injection All animals received a subcutaneous injection of AOM in saline (Sigma Chemical Co) equivalent to 16 mg of AOM per kilogram of body weight at 7 and 8 weeks of age, according to the standard protocol. One group of rats received saline injections and served as the negative controls. The rats were killed at 17 weeks of age using carbon dioxide euthanasia at the end of a feeding period after an overnight fast. 2.4. 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). 2.5. Cecal weight, cecal pH The cecum from each rat was excised, weighed, and split open, and the pH of the cecal content was measured. 2.6. Colon sample collection The colons were removed and flushed with potassium phosphate buffer (0.1 mol, pH 7.2). 2.7. Counting the ACF Each colon was split open longitudinally and placed onto a filter paper, with the luminal surface open and exposed. The secured colons were fixed overnight using 10% (volume per volume) buffered formalin. Each fixed colon was cut into a proximal and a distal portion of equal length, and each portion was further cut into 2-cm-long segments and scored for ACF, as described by Bird [17]. 2.8. Statistical analysis Values are given as mean F SEM. Data were analyzed using the SAS [24] statistical program (Statistical Analysis Systems, Inc, Cary, NC) by analysis of variance, and means
M. Verghese et al. / Nutrition Research 25 (2005) 859 – 868
863
were separated using the Tukey studentized range test. Differences were considered significant at P b .05 unless otherwise indicated. 3. Results 3.1. Feed intake and body weight Body weight gains of the rats were slightly lower, although not statistically significant in the rats fed Raftiline HP, Raftiline ST, and Raftilose compared with the groups fed the (AIN-93G) control and maltodextrin, Raftilose Synergy1, and Mix (Table 2). The differences in mean daily feed intake were not statistically significant for the rats fed the control and the experimental diets (Table 2), although the rats fed the diets with added inulin seemed to consume more diet. 3.2. Cecal weight and cecal pH Cecal pH was dramatically lowered by the diets containing Raftiline HP, Raftiline ST, Raftilose, Raftilose Synergy1, and Mix compared with the control and maltodextrin diets ( P b .05), and cecal weights were higher in all the other prebiotic carbohydrate groups compared with the control and maltodextrin group. An increase in cecal weight and decrease in cecal pH were expected (Table 2). The greatest increase in cecal weight was seen in the Mix, and the greatest decrease in cecal pH was seen in the group fed Raftiline HP (Table 2). 3.3. Diarrheal index Diarrheal index in the rats was measured. The first sign of diarrhea in the groups (Raftiline HP and Raftiline ST) appeared immediately after the second week of feeding and peaked at the third week, followed by recovery from diarrhea by the end of the fourth week of feeding. Stool consistency was firm (pelleted) throughout the study, with no visible differences noted among the groups. There were no signs of diarrhea in the other groups (data not shown). 3.4. Aberrant crypt foci The rats receiving saline injection and fed the control diet showed no evidence of ACF formation in the colon (data not shown). The results of this study demonstrated that the rats Table 2 Effect of dietary prebiotic carbohydrates on weight gain, feed intake, and cecal weight of rats Group
Weight gain (g/13 wk)
Feed intake (g/d)
Cecal weight (g)
Cecal pH
Control Maltodextrin Raftiline HP Raftiline ST Raftilose Raftilose Synergy1 Mix
236 230 220 224 229 251 260
17.0 F 17.2 F 19.1 F 18.7 F 19.2 F 19.2 F 19.1 F
0.80 F 0.02c 0.76 F 0.08c 3.16 F 0.09a 2.87 F 0.12b 2.68 F 0.19b 3.30 F 1.04a 3.86 F 1.11a
7.36 F 0.08a 7.40 F 0.10a 5.70 F 1.24c 5.96 F 1.46b 6.02 F 1.92b 6.06 F 0.12b 5.98 F 0.48b
F F F F F F F
2.92 2.04 1.98 1.64 1.45 4.26 4.01
0.39 1.56 1.08 2.02 2.14 1.16 1.08
a-c Values (mean F SEM, n = 10 per group) in columns with different superscripts are significantly different (Tukey test, P b .05).
864
M. Verghese et al. / Nutrition Research 25 (2005) 859 – 868
Fig. 1. Effect of dietary prebiotic carbohydrates on the number of colonic ACFs in rats.
fed Mix had the highest reduction of AOM-induced colonic ACF in Fisher 344 male rats compared with the control group as well as other prebiotic carbohydrate treatments. Azoxymethane treatments induced an average of 155.4 ACF per colon in the rats fed the control diet (Fig. 1). Aberrant crypt foci were predominantly observed in the distal colon (114.2). The highest reduction was seen with feeding Mix where there was a reduction of 62.1% in the proximal and 64.1% in the distal colon as compared with the rats fed the control diet. An overall reduction of 63.9% was observed in this group. A similar reduction (52.2%) was seen in the rats fed Raftilose Synergy1, with a reduction of 54.3% in the proximal and 50.8% in the distal colon as compared with the group fed the control diet. Feeding Raftiline HP, Raftiline ST, and Raftilose showed 66.7%, 64.4%, and 37.9% of reduction, respectively, in the ACF in the proximal colon; 38.5%, 17.8%, and 19.3% of reduction, respectively, in ACF in the distal colon; and a total of 46.3%, 29.6%, and 24.8% reduction in total ACF, respectively, in the total colon, compared with the control. The number of ACF in the distal colon was higher than in the proximal colon. The distal end had significantly ( P b .05) higher number of crypts than the proximal end when compared using Tukey studentized range test. These data are consistent with earlier reports that the incidence of colon cancer is significantly higher in humans in the distal end rather than in the proximal colon in humans. The administration of maltodextrin in the diet induced similar number of ACF as the rats fed the control diet (AIN-93G). Mix showed a higher reduction in AOM-induced ACF in Table 3 Effect of dietary prebiotic carbohydrates on number of total crypts in colon of rats Group
Proximal
Distal
Control Maltodextrin Raftiline HP Raftiline ST Raftilose Raftilose Synergy1 Mix
132.08 F 2.25a 130.38 F 3.57a 42.60 F 3.76d 47.50 F 1.86c,d 92.00 F 4.56b 48.26 F 0.46d 40.18 F 3.12d
426.75 380.25 249.80 323.80 307.10 190.39 135.18
Total F F F F F F F
14.89a 13.80b 5.29d 5.02c 14.45c 6.40e 4.14e
558.83 510.63 292.60 371.30 399.10 238.25 175.36
F F F F F F F
14.52a 13.34a 5.70c 3.68b 17.21b 9.98d 8.02d
a-e Values (mean F SEM, n = 10 per group) in columns with different superscripts are significantly different (Tukey test, P b .05).
M. Verghese et al. / Nutrition Research 25 (2005) 859 – 868
865
Fig. 2. Effect of dietary prebiotic carbohydrates on the number of colonic aberrant crypts/focus in rats.
Fisher 344 male rats than the groups fed Raftilose Synergy1, Raftiline HP, Raftiline ST, or Raftilose P95. 3.5. Total crypts A similar total reduction (57.4% and 68.6%) was seen in the rats fed Raftilose Synergy1 and Mix, with a reduction of 63.5% and 69.6%, respectively, in the proximal, and 55.4% and 68.3%, respectively, in the distal colon, as compared with the group fed the control diet. Feeding Raftiline HP, Raftiline ST, and Raftilose P95 showed 67.7%, 64.0%, and 30.3% of reduction, respectively, in the crypts in the proximal colon; 41.5%, 24.1%, and 28.0% of reduction, respectively, in the crypts in the distal colon; and a total of 47.6%, 33.6%, and 28.6% reduction in total crypts, respectively, in the total colon, compared with the control (Table 3). Consumption of diets containing Raftilose Synergy1, Raftiline ST, and Raftiline HP showed a reduction of total colonic crypts by 52.2%, 29.6%, and 46.3%, respectively, as compared with the control diet. The total number of foci containing 1, 2, 3, 4, and 5 or more crypts was counted in the distal and proximal regions (Fig. 2). Foci with 3, 4, and 5 or more crypts were significantly lower ( P b .05) in rats fed Raftilose Synergy1 and Mix, although foci with 1 and 2 crypts did not differ much among dietary groups. 4. Discussion In this study, we evaluated the ability of inulin-derived test compounds to reduce the development of AOM-induced foci in the colon of male Fisher 344 rats as a screening assay for these potential chemopreventive agents. The dietary test materials studied were maltodextrin to control for glucose vs fructose polymers, viz, Raftilose, Raftiline HP, Raftiline ST, Raftilose Synergy1, and Mix to examine the suppressive effects of nondigestible carbohydrates of different chain lengths. An increase in cecal weight and decrease of the pH of the cecal contents were demonstrated for all the chicory inulin products examined. In the experimental groups, the cecal weight increased and the pH of the cecal contents decrease correlated with the average
866
M. Verghese et al. / Nutrition Research 25 (2005) 859 – 868
chain length of the inulin products, with the greatest effect being seen for Mix, which is a 1:2 mixture of long-chain and short-chain polymers. The increase in cecal weight due to ingestion of prebiotic carbohydrates may result from SCFAs promoting cecal growth. The decrease in cecal pH may be caused by the greater level of SCFA production [25]. A significant increase in cecal weight and a significant decrease in cecal pH have also been reported by our laboratory and other researchers [9,12,13] after feeding diets containing 5% and 10% inulin. It was suggested that consumption of inulin was associated with potentially beneficial changes in cecal physiology and bacterial metabolic activity in relation to the incidence of preneoplastic lesions and tumor risk in the colon. A reduction in cecal pH and a reduction in fecal pH (data not shown) was seen in this study. Campbell et al [25] suggested that a reduction in fecal pH is a possible factor in the suppression of colon tumorigenesis. Many researchers have demonstrated a bifidogenic effect of inulin and oligofructose [14,15,26,27]. The metabolites produced by the bifidobacteria may affect the mixed-function oxidases, ornithine decarboxylase, and ras p21 expression [7]. The inhibitory effect of bifidobacteria on AOM-induced ACF has been reported by our laboratory [28] and Kulkarni and Reddy [29]. The magnitude of the difference from the control group of Mix was greater than that of Raftilose Synergy1. The results illustrate a correlation between the reduction in the numbers of ACF in the rat colon and increase in chain length of the dietary inulin preparations. This might be related to the fact that longchain inulin preparations ferment more slowly than short ones (in vitro 5 hours for short chains vs 15 hours for long chains with fecal slurry). The percentage reduction in the numbers of ACF increases in the order Raftilose (DPav 4) b Raftiline ST (DPav 10) b Raftiline HP (DPav 25) b Raftilose Synergy1 (1:1 mix of DPav 4/DPav 25) b Mix (1:2 mix of DPav 4/DPav 25). It is possible that the short-chain fraction in Synergy1 is able to modify the composition of the intestinal flora and that the long chains can maintain the metabolism of the bimprovedQ flora. As a consequence, it can exert its beneficial effects for a longer period. The effect with Mix is more pronounced than with Raftilose Synergy1, meaning that the optimal combination of chain lengths may not have been found. Presumably, the optimal combination will be a product containing a minimum effective level of short chains to modify the microflora. These and other anticancer properties of dietary inulin and oligofructose, and their combinations justify further human dietary intervention follow-up studies. Acknowledgment This study was supported by ORAFTI, Tienen, Belgium, and The Agricultural Experiment Station, Alabama A&M University, Normal, Ala. References [1] Parker SL, Tong T, Bolden S, Wings PA. Cancer statistics. CA Cancer J Clin 1997;47(Suppl 1):5 - 27. [2] Potter JD. Risk factors for colon neoplasia. Epidemiology and biology. Eur J Cancer 1995;23:1210 - 20. [3] Agarwal A, Shen H, Agarwal S, Rao AV. Lycopene content of tomato products: its stability, bioavailability and in vivo antioxidant properties. J Med Food 2001;4(1):9 - 15.
M. Verghese et al. / Nutrition Research 25 (2005) 859 – 868
867
[4] Franceschi S, Levi F, La Vecchia C. Epidemiology of gastric cancer in Europe. Eur J Cancer Prev 1994;2(Suppl 2):5 - 10. [5] Gerster H. The potential role of lycopene for human health. J Am Coll Nutr 1997;16(2):109 - 26. [6] Giovannucci E, Ascherio A, Rimm EB, Stampfer MJ, Colditz GA, Willett WC. Intake of carotenoids and retinol in relation to risk of prostate cancer. J Natl Cancer Inst 1995;87:1767 - 76. [7] Reddy BS. Possible mechanisms by which pro- and prebiotics influence colon carcinogenesis and tumor growth. J Nutr 1999;129:1478S - 82S. [8] Reddy BS, Hamid R, Rao CV. Effect of dietary oligofructose and inulin on colonic preneoplastic aberrant crypt foci inhibition. Carcinogenesis 1997;18:1371 - 4. [9] Rowland IR, Rumney CJ, Coutts JT, Lievense L. Effects of Bifidobacterium longum and inulin on gut bacterial metabolism and carcinogen induced aberrant crypt foci in rats. Carcinogenesis 1998;19: 281 - 5. [10] Taper H, Delzenne N, Roberfroid MB. Growth inhibition of transplantable mouse tumours by non digestible carbohydrates. Int J Cancer 1997;71:1109 - 12. [11] Taper H, Roberfroid MB. Influence of inulin and oligofructose on breast cancer and tumor growth. J Nutr 1999;129:1488S - 91S. [12] Verghese M, Rao DR, Chawan CB, Shackelford L. Dietary inulin suppresses azoxymethane-induced preneoplastic aberrant crypt foci in mature Fisher 344 rats. J Nutr 2002;132:2809 - 13. [13] Verghese M, Rao DR, Chawan CB, Williams LL, Shackelford L. Dietary inulin suppresses azoxymethane-induced aberrant crypt foci and colon tumors at promotion stage in Fisher 344 rats. J Nutr 2002;132:2804 - 8. [14] Hidaka H, Eida T, Takizawa T, Tokunaga T, Tashiro Y. Effects of fructooligosaccharides on intestinal flora and human health. Bifidibact Microflora 1986;5:37. [15] Mitsuoka T, Hidaka H, Eida T. Effect of fructo-oligosaccharides on intestinal microflora. Nahrung 1987;31:427 - 36. [16] Van Loo J, Jonkers N. Evaluation in human volunteers of the potential anticarcinogenic activities of novel nutritional concepts: prebiotics probiotics and synbiotics (the SYNCAN project). Nutr Metab Cardiovasc Dis 2001;11:87 - 93. [17] Bird RP. Observation and quantification of aberrant crypt foci in murine colon treated with a colon carcinogen: preliminary findings. Cancer Lett 1987;37:147 - 51. [18] Bird RP, Good CK. The significance of aberrant crypt foci in understanding the pathogenesis of colon cancer. Toxicol Lett 2000;112-113:395 - 402. [19] Wargovich MJ, Jimenez A, McKee K, Steele VE, Velasco M, Woods J, et al. Efficacy of potential chemopreventive agents on rat colon aberrant crypt formation and progression. Carcinogenesis 2000;21(6):1149 - 55. [20] Siu WY, Arooz T, Poon RY. Differential responses of proliferating versus quiescent cells to adriamycin. Exp Cell Res 1999;250(1):131 - 41. [21] Pretlow TP, O’Riordan MA, Pretlow TG, Stellato TA. Aberrant crypts in human colonic mucosa: putative preneoplastic lesions. J Cell Biochem 1992;16G(Suppl):55 - 62. [22] Pretlow TP, O’Riordan MA, Somich GA, Pretlow TG. Aberrant crypts correlate with tumor incidence in Fisher344 rats treated with azoxymethane and phytate. Carcinogenesis 1992;13:1509 - 12. [23] Reeves PG, Nielsen FH, Fahey Jr GC. 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 1993;123:1939 - 51. [24] SAS. Statistical Analysis Systems, Inc Cary (NC); 1997. [25] Campbell JM, Fahey GC, Wolf BW. Selected indigestible oligosaccharides affect large bowel mass, cecal and fecal short-chain fatty acids, pH and microflora in rats. J Nutr 1997;127:30 - 136. [26] Roberfroid MA. A functional food — chicory fructo-oligosaccharide: a colonic food with prebiotic activity. World Ingred 1995;42 - 4. [27] Gibson GR, Roberfroid MB. Dietary modulation of the human colonic microbiota — introducing the concept of prebiotics. J Nutr 1995;125:1401 - 12.
868
M. Verghese et al. / Nutrition Research 25 (2005) 859 – 868
[28] Challa A, Rao DR, Chawan CB, Shackelford L. Bifidobacterium longum and lactulose suppress azoxymethane-induced colonic aberrant crypt foci in rats. Carcinogenesis 1997;18:517 - 21. [29] Kulkarni N, Reddy BS. Inhibitory effect of Bifidobacterium longum cultures on azoxymethane-induced aberrant crypt foci formation and fecal bacterial b-glucuronidase. Proc Soc Exp Biol Med 1994; 207:278 - 83.
Inhibitory effects of nondigestible carbohydrates of different chain lengths on azoxymethane-induced aberrant crypt foci in Fisher 344 rats Martha VergheseT, Lloyd T. Walker, Louis Shackelford, Chandramohan B. Chawan Nutrition and Carcinogenesis Laboratory, Department of Food and Animal Sciences, Alabama A&M University, P.O. Box 1628, Normal, AL 35762, USA Received 12 September 2004; revised 3 August 2005; accepted 1 September 2005
Abstract Colon cancer is one of the leading causes of cancer morbidity and mortality in Western countries. The objective of this study was to elucidate the effect of prebiotic carbohydrates of different chain lengths on azoxymethane-induced aberrant crypt foci in Fisher 344 male rats. After an acclimatization period of 1 week, 70 male weanling rats were divided into 7 groups and fed AIN-93G (Control) and 6 experimental diets that contained control + (maltodextrin; Raftiline HP, Raftiline ST, Raftilose P95, Raftilose Synergy1, and Mix; ORAFTI, Tienen, Belgium). All the rats received 16 mg/kg body weight of azoxymethane dissolved in saline subcutaneous at 7 and 8 weeks of age. The rats continued to receive the assigned diets until killed by carbon dioxide asphyxiation at 17 weeks of age. There was a significant ( P b .05) increase in cecal weight and a decrease in cecal pH in rats fed prebiotic carbohydrates. The highest reduction of colonic aberrant crypt foci, both in total number as well as crypt, multiplicity was seen in the group fed Mix (63.9%). Consumption of diets containing Raftilose Synergy1, Raftiline ST, and Raftiline HP showed a reduction of total colonic crypts by 52.2%, 29.6%, and 46.3%, respectively, as compared with the control diet. D 2005 Elsevier Inc. All rights reserved. Keywords: Rat; ACFs, aberrant crypt foci; AOM, azoxymethane; Fructo-oligosaccharides; SCFAs, short-chain fatty acids
T Corresponding author. E-mail address: [email protected] (M. Verghese). 0271-5317/$ – see front matter D 2005 Elsevier Inc. All rights reserved. doi:10.1016/j.nutres.2005.09.007
860
M. Verghese et al. / Nutrition Research 25 (2005) 859 – 868
1. Introduction Colon cancer is the second leading cause of cancer-related deaths. Although cancer of the colon is widely believed to be more prevalent in men, it is actually more common in women [1]. Sometimes referred to as colorectal cancer (cancer of the colon and rectum), it is predicted to be responsible for about 56 000 deaths during the year 1999. Several factors are believed to be involved in the generation of cancer. According to the American Institute for Cancer Research, some of the most common causes of cancer include heredity, tobacco use, and high consumption of alcohol. The link between diet, nutrition, and the risk for cancer is well established [2]. Studies have shown that about 50% of all cancers are the result of poor diet [3]. Diets rich in fat and poor in fiber can increase the incidence of various forms of cancer. For this reason, nutritionists recommend diets rich in largely unprocessed fruits and vegetables, which are generally low in fat and represent the source of naturally occurring dietary fiber and cancerfighting nutrients such as vitamin A, vitamin C, and carotenoids. These substances are very effective against cancer of the lung, breast, oral cavity, esophagus, stomach, pancreas, cervix, and prostate, especially the colon and rectum [4-6]. Recent research has revealed that certain classes of nondigestible carbohydrates such as inulin, which is extracted from chicory root by processes that are similar to beet sugar processing, have been shown to be chemopreventive in various experimental models [7-13]. These carbohydrates that are not digested or absorbed in the stomach or small intestine arrive almost quantitatively in the colon where they are fermented selectively by certain groups of bacteria (bifidobacteria, lactobacilli, and others). This increased bacterial metabolic activity is thought to be the basis of the anticancer properties of these prebiotic carbohydrates. The effects may be realized through increased production of short-chain fatty acids (SCFAs) through bacterial cell wall interactions with the host (immunologic) and/or reduced production of genotoxic and putrefactive compounds in the intestinal lumen. Fructooligosaccharides have been shown to be indigestible by enzymes in the human small intestine but are fermented extensively by large-bowel microflora [14,15]. There is preliminary evidence of the inhibitory effects of inulin in experimental animals [8,12,13,16]. A number of natural and synthetic compounds are being studied as potentially chemopreventive for colon cancer using the induction of aberrant crypt foci (ACFs) as the primary end point. Aberrant crypt foci are preneoplastic lesions in rat colon, and they are induced by all colon carcinogens [17,18]. Aberrant crypt foci are preneoplastic lesions found in most colon cancers [19]. Aberrant crypt foci are characterized by 1 or more crypts that appear as a single focus but are larger than normal crypts, have thickened epithelia, possess altered luminal openings, have an increased pericryptal area between them and normal crypts, and appear elevated compared with normal crypts when viewed under a microscope [20,21]. Each ACF contains numerous crypts per focus that multiply with time after treatment with a carcinogen and can be observed as early as 2 weeks after injection of the carcinogen [22]. These ACF can develop into polyps and eventually into colon cancer. Foci of aberrant crypts in rat colon are putative cancerous lesions that have been proposed as biomarkers for short-term assays for testing chemical carcinogens and chemopreventive
M. Verghese et al. / Nutrition Research 25 (2005) 859 – 868
861
agents. Here, the ability of test compounds (maltodextrin; Raftiline HP, Raftiline ST, Raftilose, Raftilose Synergy1, and Mix; ORAFTI Tienen, Belgium) to reduce the development of azoxymethane (AOM)–induced foci in the colon of male Fisher 344 rats was evaluated as a screening assay for these potential chemopreventive agents. This study was designed to determine whether (1) dietary inulin preparations of different chain lengths can reduce AOM-induced ACF in the rat colon and (2) the chemopreventive effect can be increased by combining inulin fractions (Raftiline HP and oligofructose). 2. Methods and materials 2.1. Animals, housing, and diets After 1 week of acclimatization, male Fisher 344 weanling rats were divided into 8 groups of 10 rats each and assigned the AIN-93G (Control-C) and experimental diets for 13 weeks in ACF study. Temperature and relative humidity were maintained at 218C F 18C and 50%, respectively. Light and dark cycles were for 12 hours each. Feed and water were provided ad libitum. Body weights and feed intakes were recorded weekly. The diets were prepared fresh every week and stored at 48C. Diets (Table 1) were formulated based on AIN-93G diets [23], and the ingredients were obtained from ICN Biochemicals, Costa Mesa, Calif. All the protocols involving rats have been approved by the Institutional Animal Care and Use Committee of Alabama A&M University. Prebiotic carbohydrates were obtained from ORAFTI. All other chemicals were obtained from Sigma Chemical Co (St Louis, Mo). 2.2. Prebiotic carbohydrates Various fractions of chicory inulin were used in this study. 1. Raftiline ST or native chicory inulin is a polydisperse mixture of linear molecules, all with the same basic chemical structure, which is symbolized as G-Fn Table 1 Composition of the diets fed to rats Ingredients (g/kg)
Control
Prebiotic carbohydratesa
Corn starch Prebiotic carbohydratesa Casein (85% protein) Dextrin Sucrose Fiberb Oil (soybean, no additives) Mineral mix (AIN-93G MX) Vitamin mix (AIN-93 VX) Cystine Choline bitartrate (41.1% choline)
397.5 0 200 132 100 50 70 35 10 3 2.5
297.5 100 200 132 100 50 70 35 10 3 2.5
MX = mineral mix; VX = vitamin mix. a Prebiotic carbohydrates: maltodextrin, Raftiline HP, Raftiline ST, Raftilose Synergy 1, and Mix. b Solk-FlocR, 200 FCC (FS&D, St Louis, Mo) or equivalent.
862
2. 3.
4. 5.
M. Verghese et al. / Nutrition Research 25 (2005) 859 – 868
(G indicates glucosyl moiety; F, fructosyl moiety; and n, number of fructose units linked together through b(2-1) bonds). The degree of polymerization (DP) of native chicory inulin ranges between 3 and 65, with an average degree of polymerization (DPav) of 10. Raftiline HP is inulin from which the lower DP fraction has been removed, resulting in a DPav of about 25 (also called HP-inulin). Raftilose P95 is a short-chain fraction (also called oligofructose) that is obtained by partial enzymatic hydrolysis of native chicory inulin. It is composed of linear G-Fn and F-Fn chains, with DP ranging from 2 to 8 (DPav is about 4). Raftilose Synergy1 is a 1:1 (weight per weight) co-spray-dried mixture of oligofructose and Raftiline HP. Mix is an experimental product that is composed of 1:2 (weight per weight) oligofructose-Raftiline HP.
2.3. Carcinogen injection All animals received a subcutaneous injection of AOM in saline (Sigma Chemical Co) equivalent to 16 mg of AOM per kilogram of body weight at 7 and 8 weeks of age, according to the standard protocol. One group of rats received saline injections and served as the negative controls. The rats were killed at 17 weeks of age using carbon dioxide euthanasia at the end of a feeding period after an overnight fast. 2.4. 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). 2.5. Cecal weight, cecal pH The cecum from each rat was excised, weighed, and split open, and the pH of the cecal content was measured. 2.6. Colon sample collection The colons were removed and flushed with potassium phosphate buffer (0.1 mol, pH 7.2). 2.7. Counting the ACF Each colon was split open longitudinally and placed onto a filter paper, with the luminal surface open and exposed. The secured colons were fixed overnight using 10% (volume per volume) buffered formalin. Each fixed colon was cut into a proximal and a distal portion of equal length, and each portion was further cut into 2-cm-long segments and scored for ACF, as described by Bird [17]. 2.8. Statistical analysis Values are given as mean F SEM. Data were analyzed using the SAS [24] statistical program (Statistical Analysis Systems, Inc, Cary, NC) by analysis of variance, and means
M. Verghese et al. / Nutrition Research 25 (2005) 859 – 868
863
were separated using the Tukey studentized range test. Differences were considered significant at P b .05 unless otherwise indicated. 3. Results 3.1. Feed intake and body weight Body weight gains of the rats were slightly lower, although not statistically significant in the rats fed Raftiline HP, Raftiline ST, and Raftilose compared with the groups fed the (AIN-93G) control and maltodextrin, Raftilose Synergy1, and Mix (Table 2). The differences in mean daily feed intake were not statistically significant for the rats fed the control and the experimental diets (Table 2), although the rats fed the diets with added inulin seemed to consume more diet. 3.2. Cecal weight and cecal pH Cecal pH was dramatically lowered by the diets containing Raftiline HP, Raftiline ST, Raftilose, Raftilose Synergy1, and Mix compared with the control and maltodextrin diets ( P b .05), and cecal weights were higher in all the other prebiotic carbohydrate groups compared with the control and maltodextrin group. An increase in cecal weight and decrease in cecal pH were expected (Table 2). The greatest increase in cecal weight was seen in the Mix, and the greatest decrease in cecal pH was seen in the group fed Raftiline HP (Table 2). 3.3. Diarrheal index Diarrheal index in the rats was measured. The first sign of diarrhea in the groups (Raftiline HP and Raftiline ST) appeared immediately after the second week of feeding and peaked at the third week, followed by recovery from diarrhea by the end of the fourth week of feeding. Stool consistency was firm (pelleted) throughout the study, with no visible differences noted among the groups. There were no signs of diarrhea in the other groups (data not shown). 3.4. Aberrant crypt foci The rats receiving saline injection and fed the control diet showed no evidence of ACF formation in the colon (data not shown). The results of this study demonstrated that the rats Table 2 Effect of dietary prebiotic carbohydrates on weight gain, feed intake, and cecal weight of rats Group
Weight gain (g/13 wk)
Feed intake (g/d)
Cecal weight (g)
Cecal pH
Control Maltodextrin Raftiline HP Raftiline ST Raftilose Raftilose Synergy1 Mix
236 230 220 224 229 251 260
17.0 F 17.2 F 19.1 F 18.7 F 19.2 F 19.2 F 19.1 F
0.80 F 0.02c 0.76 F 0.08c 3.16 F 0.09a 2.87 F 0.12b 2.68 F 0.19b 3.30 F 1.04a 3.86 F 1.11a
7.36 F 0.08a 7.40 F 0.10a 5.70 F 1.24c 5.96 F 1.46b 6.02 F 1.92b 6.06 F 0.12b 5.98 F 0.48b
F F F F F F F
2.92 2.04 1.98 1.64 1.45 4.26 4.01
0.39 1.56 1.08 2.02 2.14 1.16 1.08
a-c Values (mean F SEM, n = 10 per group) in columns with different superscripts are significantly different (Tukey test, P b .05).
864
M. Verghese et al. / Nutrition Research 25 (2005) 859 – 868
Fig. 1. Effect of dietary prebiotic carbohydrates on the number of colonic ACFs in rats.
fed Mix had the highest reduction of AOM-induced colonic ACF in Fisher 344 male rats compared with the control group as well as other prebiotic carbohydrate treatments. Azoxymethane treatments induced an average of 155.4 ACF per colon in the rats fed the control diet (Fig. 1). Aberrant crypt foci were predominantly observed in the distal colon (114.2). The highest reduction was seen with feeding Mix where there was a reduction of 62.1% in the proximal and 64.1% in the distal colon as compared with the rats fed the control diet. An overall reduction of 63.9% was observed in this group. A similar reduction (52.2%) was seen in the rats fed Raftilose Synergy1, with a reduction of 54.3% in the proximal and 50.8% in the distal colon as compared with the group fed the control diet. Feeding Raftiline HP, Raftiline ST, and Raftilose showed 66.7%, 64.4%, and 37.9% of reduction, respectively, in the ACF in the proximal colon; 38.5%, 17.8%, and 19.3% of reduction, respectively, in ACF in the distal colon; and a total of 46.3%, 29.6%, and 24.8% reduction in total ACF, respectively, in the total colon, compared with the control. The number of ACF in the distal colon was higher than in the proximal colon. The distal end had significantly ( P b .05) higher number of crypts than the proximal end when compared using Tukey studentized range test. These data are consistent with earlier reports that the incidence of colon cancer is significantly higher in humans in the distal end rather than in the proximal colon in humans. The administration of maltodextrin in the diet induced similar number of ACF as the rats fed the control diet (AIN-93G). Mix showed a higher reduction in AOM-induced ACF in Table 3 Effect of dietary prebiotic carbohydrates on number of total crypts in colon of rats Group
Proximal
Distal
Control Maltodextrin Raftiline HP Raftiline ST Raftilose Raftilose Synergy1 Mix
132.08 F 2.25a 130.38 F 3.57a 42.60 F 3.76d 47.50 F 1.86c,d 92.00 F 4.56b 48.26 F 0.46d 40.18 F 3.12d
426.75 380.25 249.80 323.80 307.10 190.39 135.18
Total F F F F F F F
14.89a 13.80b 5.29d 5.02c 14.45c 6.40e 4.14e
558.83 510.63 292.60 371.30 399.10 238.25 175.36
F F F F F F F
14.52a 13.34a 5.70c 3.68b 17.21b 9.98d 8.02d
a-e Values (mean F SEM, n = 10 per group) in columns with different superscripts are significantly different (Tukey test, P b .05).
M. Verghese et al. / Nutrition Research 25 (2005) 859 – 868
865
Fig. 2. Effect of dietary prebiotic carbohydrates on the number of colonic aberrant crypts/focus in rats.
Fisher 344 male rats than the groups fed Raftilose Synergy1, Raftiline HP, Raftiline ST, or Raftilose P95. 3.5. Total crypts A similar total reduction (57.4% and 68.6%) was seen in the rats fed Raftilose Synergy1 and Mix, with a reduction of 63.5% and 69.6%, respectively, in the proximal, and 55.4% and 68.3%, respectively, in the distal colon, as compared with the group fed the control diet. Feeding Raftiline HP, Raftiline ST, and Raftilose P95 showed 67.7%, 64.0%, and 30.3% of reduction, respectively, in the crypts in the proximal colon; 41.5%, 24.1%, and 28.0% of reduction, respectively, in the crypts in the distal colon; and a total of 47.6%, 33.6%, and 28.6% reduction in total crypts, respectively, in the total colon, compared with the control (Table 3). Consumption of diets containing Raftilose Synergy1, Raftiline ST, and Raftiline HP showed a reduction of total colonic crypts by 52.2%, 29.6%, and 46.3%, respectively, as compared with the control diet. The total number of foci containing 1, 2, 3, 4, and 5 or more crypts was counted in the distal and proximal regions (Fig. 2). Foci with 3, 4, and 5 or more crypts were significantly lower ( P b .05) in rats fed Raftilose Synergy1 and Mix, although foci with 1 and 2 crypts did not differ much among dietary groups. 4. Discussion In this study, we evaluated the ability of inulin-derived test compounds to reduce the development of AOM-induced foci in the colon of male Fisher 344 rats as a screening assay for these potential chemopreventive agents. The dietary test materials studied were maltodextrin to control for glucose vs fructose polymers, viz, Raftilose, Raftiline HP, Raftiline ST, Raftilose Synergy1, and Mix to examine the suppressive effects of nondigestible carbohydrates of different chain lengths. An increase in cecal weight and decrease of the pH of the cecal contents were demonstrated for all the chicory inulin products examined. In the experimental groups, the cecal weight increased and the pH of the cecal contents decrease correlated with the average
866
M. Verghese et al. / Nutrition Research 25 (2005) 859 – 868
chain length of the inulin products, with the greatest effect being seen for Mix, which is a 1:2 mixture of long-chain and short-chain polymers. The increase in cecal weight due to ingestion of prebiotic carbohydrates may result from SCFAs promoting cecal growth. The decrease in cecal pH may be caused by the greater level of SCFA production [25]. A significant increase in cecal weight and a significant decrease in cecal pH have also been reported by our laboratory and other researchers [9,12,13] after feeding diets containing 5% and 10% inulin. It was suggested that consumption of inulin was associated with potentially beneficial changes in cecal physiology and bacterial metabolic activity in relation to the incidence of preneoplastic lesions and tumor risk in the colon. A reduction in cecal pH and a reduction in fecal pH (data not shown) was seen in this study. Campbell et al [25] suggested that a reduction in fecal pH is a possible factor in the suppression of colon tumorigenesis. Many researchers have demonstrated a bifidogenic effect of inulin and oligofructose [14,15,26,27]. The metabolites produced by the bifidobacteria may affect the mixed-function oxidases, ornithine decarboxylase, and ras p21 expression [7]. The inhibitory effect of bifidobacteria on AOM-induced ACF has been reported by our laboratory [28] and Kulkarni and Reddy [29]. The magnitude of the difference from the control group of Mix was greater than that of Raftilose Synergy1. The results illustrate a correlation between the reduction in the numbers of ACF in the rat colon and increase in chain length of the dietary inulin preparations. This might be related to the fact that longchain inulin preparations ferment more slowly than short ones (in vitro 5 hours for short chains vs 15 hours for long chains with fecal slurry). The percentage reduction in the numbers of ACF increases in the order Raftilose (DPav 4) b Raftiline ST (DPav 10) b Raftiline HP (DPav 25) b Raftilose Synergy1 (1:1 mix of DPav 4/DPav 25) b Mix (1:2 mix of DPav 4/DPav 25). It is possible that the short-chain fraction in Synergy1 is able to modify the composition of the intestinal flora and that the long chains can maintain the metabolism of the bimprovedQ flora. As a consequence, it can exert its beneficial effects for a longer period. The effect with Mix is more pronounced than with Raftilose Synergy1, meaning that the optimal combination of chain lengths may not have been found. Presumably, the optimal combination will be a product containing a minimum effective level of short chains to modify the microflora. These and other anticancer properties of dietary inulin and oligofructose, and their combinations justify further human dietary intervention follow-up studies. Acknowledgment This study was supported by ORAFTI, Tienen, Belgium, and The Agricultural Experiment Station, Alabama A&M University, Normal, Ala. References [1] Parker SL, Tong T, Bolden S, Wings PA. Cancer statistics. CA Cancer J Clin 1997;47(Suppl 1):5 - 27. [2] Potter JD. Risk factors for colon neoplasia. Epidemiology and biology. Eur J Cancer 1995;23:1210 - 20. [3] Agarwal A, Shen H, Agarwal S, Rao AV. Lycopene content of tomato products: its stability, bioavailability and in vivo antioxidant properties. J Med Food 2001;4(1):9 - 15.
M. Verghese et al. / Nutrition Research 25 (2005) 859 – 868
867
[4] Franceschi S, Levi F, La Vecchia C. Epidemiology of gastric cancer in Europe. Eur J Cancer Prev 1994;2(Suppl 2):5 - 10. [5] Gerster H. The potential role of lycopene for human health. J Am Coll Nutr 1997;16(2):109 - 26. [6] Giovannucci E, Ascherio A, Rimm EB, Stampfer MJ, Colditz GA, Willett WC. Intake of carotenoids and retinol in relation to risk of prostate cancer. J Natl Cancer Inst 1995;87:1767 - 76. [7] Reddy BS. Possible mechanisms by which pro- and prebiotics influence colon carcinogenesis and tumor growth. J Nutr 1999;129:1478S - 82S. [8] Reddy BS, Hamid R, Rao CV. Effect of dietary oligofructose and inulin on colonic preneoplastic aberrant crypt foci inhibition. Carcinogenesis 1997;18:1371 - 4. [9] Rowland IR, Rumney CJ, Coutts JT, Lievense L. Effects of Bifidobacterium longum and inulin on gut bacterial metabolism and carcinogen induced aberrant crypt foci in rats. Carcinogenesis 1998;19: 281 - 5. [10] Taper H, Delzenne N, Roberfroid MB. Growth inhibition of transplantable mouse tumours by non digestible carbohydrates. Int J Cancer 1997;71:1109 - 12. [11] Taper H, Roberfroid MB. Influence of inulin and oligofructose on breast cancer and tumor growth. J Nutr 1999;129:1488S - 91S. [12] Verghese M, Rao DR, Chawan CB, Shackelford L. Dietary inulin suppresses azoxymethane-induced preneoplastic aberrant crypt foci in mature Fisher 344 rats. J Nutr 2002;132:2809 - 13. [13] Verghese M, Rao DR, Chawan CB, Williams LL, Shackelford L. Dietary inulin suppresses azoxymethane-induced aberrant crypt foci and colon tumors at promotion stage in Fisher 344 rats. J Nutr 2002;132:2804 - 8. [14] Hidaka H, Eida T, Takizawa T, Tokunaga T, Tashiro Y. Effects of fructooligosaccharides on intestinal flora and human health. Bifidibact Microflora 1986;5:37. [15] Mitsuoka T, Hidaka H, Eida T. Effect of fructo-oligosaccharides on intestinal microflora. Nahrung 1987;31:427 - 36. [16] Van Loo J, Jonkers N. Evaluation in human volunteers of the potential anticarcinogenic activities of novel nutritional concepts: prebiotics probiotics and synbiotics (the SYNCAN project). Nutr Metab Cardiovasc Dis 2001;11:87 - 93. [17] Bird RP. Observation and quantification of aberrant crypt foci in murine colon treated with a colon carcinogen: preliminary findings. Cancer Lett 1987;37:147 - 51. [18] Bird RP, Good CK. The significance of aberrant crypt foci in understanding the pathogenesis of colon cancer. Toxicol Lett 2000;112-113:395 - 402. [19] Wargovich MJ, Jimenez A, McKee K, Steele VE, Velasco M, Woods J, et al. Efficacy of potential chemopreventive agents on rat colon aberrant crypt formation and progression. Carcinogenesis 2000;21(6):1149 - 55. [20] Siu WY, Arooz T, Poon RY. Differential responses of proliferating versus quiescent cells to adriamycin. Exp Cell Res 1999;250(1):131 - 41. [21] Pretlow TP, O’Riordan MA, Pretlow TG, Stellato TA. Aberrant crypts in human colonic mucosa: putative preneoplastic lesions. J Cell Biochem 1992;16G(Suppl):55 - 62. [22] Pretlow TP, O’Riordan MA, Somich GA, Pretlow TG. Aberrant crypts correlate with tumor incidence in Fisher344 rats treated with azoxymethane and phytate. Carcinogenesis 1992;13:1509 - 12. [23] Reeves PG, Nielsen FH, Fahey Jr GC. 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 1993;123:1939 - 51. [24] SAS. Statistical Analysis Systems, Inc Cary (NC); 1997. [25] Campbell JM, Fahey GC, Wolf BW. Selected indigestible oligosaccharides affect large bowel mass, cecal and fecal short-chain fatty acids, pH and microflora in rats. J Nutr 1997;127:30 - 136. [26] Roberfroid MA. A functional food — chicory fructo-oligosaccharide: a colonic food with prebiotic activity. World Ingred 1995;42 - 4. [27] Gibson GR, Roberfroid MB. Dietary modulation of the human colonic microbiota — introducing the concept of prebiotics. J Nutr 1995;125:1401 - 12.
868
M. Verghese et al. / Nutrition Research 25 (2005) 859 – 868
[28] Challa A, Rao DR, Chawan CB, Shackelford L. Bifidobacterium longum and lactulose suppress azoxymethane-induced colonic aberrant crypt foci in rats. Carcinogenesis 1997;18:517 - 21. [29] Kulkarni N, Reddy BS. Inhibitory effect of Bifidobacterium longum cultures on azoxymethane-induced aberrant crypt foci formation and fecal bacterial b-glucuronidase. Proc Soc Exp Biol Med 1994; 207:278 - 83.