Effects of fiber and fat on murine proximal colonic mucosal proliferation and crypt depth

Nutrition Research 24 (2004) 901 – 908 www.elsevier.com/locate/nutres

Effects of fiber and fat on murine proximal colonic mucosal proliferation and crypt depth Marc D. Bassona,*, Cheng Fang Yuc, Ruben Gomeza, Omar Bashirb, Robert A. Goodladb a

Department of Surgery, John D. Dingell Veterans Administration Medical Center and Wayne State University, Detroit, MI 48201-1932, USA b Department of Cancer Research UK, Histopathology Unit, 44 Lincoln’s Inn Fields, London WC2A 3PX, UK c Bristol-Myers Squibb, 5 Research Pkwy, Wallingford, CT 06492, USA Received 21 November 2003; revised 24 June 2004; accepted 28 June 2004

Abstract The effects of 5 defined experimental diets and a commercial mouse diet on colonic epithelial proliferation in the C57BL/6L mouse were studied. Crypt length, mitoses per crypt, Ki-67–labeled cells per crypt, and growth fraction was assessed in colonic crypts from histologic slides. A control diet of no fiber and high fat produced the highest measures of proliferation. In comparison to the control, the no-fiber, low-fat diet and the 10% cellulose, high-fat diets showed statistically significant decreases in the measures of proliferation ( P b .05). Fiber supplementation with wheat bran or guar gum was also associated with a tendency toward decreased mucosal proliferation, but these results were not statistically significant. This study demonstrates that dietary fiber can reduce the mitogenic impact of a high-fat diet upon the colonic mucosa in mice, but that these effects may be fiber dependent. Published by Elsevier Inc. Keywords: Proliferation; Colonic; Epithelial; Low-fat diet; High-fat diet; Dietary fiber

1. Introduction Although epidemiological and experimental data suggest that dietary fiber intake affects the colonic mucosa, the nature and mechanism of these effects are unclear. Supplemental fiber effects may vary with the dose, nature, particle size, and preparation of the fiber, as well as the * Corresponding author. Marc D. Basson, Chief of Surgical Services, John D. Dingell VA Medical Center, Detroit, MI 48201-1932, USA. Tel.: +1 313 576 3598; fax: +1 313 576 1002. E-mail address: [email protected] (M.D. Basson). 0271-5317/$ – see front matter Published by Elsevier Inc. doi:10.1016/j.nutres.2004.06.009

902

M.D. Basson et al. / Nutrition Research 24 (2004) 901 – 908

dietary background upon which fiber supplementation occurs and micronutrients that may be consumed along with natural dietary fiber. We [1] recently compared the effects of dietary supplementation with 3 different fibers, cellulose, guar gum, and wheat bran, on the development of intestinal neoplasia in APC/MIN mice consuming an experimental 20% fat diet, and characterized the effects of these diets on both APC/MIN mice and the congeneic C57BL/J background strain. We also assessed the effect of reducing the fat to 5%. Although these various diets resulted in a lower incidence of colonic neoplasia than the commercial solid diet that served as an additional control, the differences in colonic neoplasia among mice consuming the various experimental diets were relatively small. However, we did note that stool bulk was substantially increased in response to the various dietary fibers. In separate investigations, we have noted that repetitive strain is trophic for intestinal epithelial cell monolayers in vitro, stimulating their proliferation in an amplitude- and frequency-dependent manner [2,3] via protein kinase C (PKC), focal adhesion kinase, and mitogen-activated protein kinase activation [4-6]. Tyrosine kinase, PKC, and extracellular signal-regulated kinase inhibitors prevent the stimulation of proliferation by repetitive strain [4,5]. In vivo, repetitive mucosal strain and pressure induced by pulsatile perfusion of defunctionalized intestinal loops in anesthetized animals results in increased mucosal tyrosine kinase activity. [7] Because fiber supplementation softens the stool and reduces luminal strain and pressure [8], we hypothesized that normal colonic mucosal proliferation might be altered by consumption of these diets even if colonic neoplastic transformation was unaffected. We therefore studied the effects of consumption of these diets on the proliferation and crypt depth of the proximal colonic mucosa in C57BL/6L mice consuming experimental diets. Mice were fed experimental diets with 20% fat from soybean oil without any fiber or supplemented by 10% cellulose, wheat bran, or guar gum. A 5% soybean oil–fat diet without fiber and a commercial mouse diet served as additional controls. Dietary consumption, animal weight gain, and stool weight were monitored in these animals. After 30 days, the right colon was resected at time of sacrifice, formalin fixed, and assessed for mitotic figures, Ki-67 immunoreactivity, crypt length, and the maximum and 50% labeling position within the crypts. 2. Methods 2.1. Animals and housing Forty eight C57BL/6L mice (male:female = 1:1) were obtained from Jackson Laboratory, Bar Harbor, Me. They were approximately 5 weeks of age and weighed 16 to 22 g at the beginning of the study. The mice were randomly divided into 6 groups of 8 animals and housed 4 to a cage with a wire drop bottom (no bedding) to minimize coprophagy and consumption of bedding. 2.2. Diets Six diets were studied, all in pelleted form. The first was the Harlan Teklad LM-485 commercial mouse chow, which contains 5.67% fat (soybean meal and soybean oil) and 4.37% crude fiber (ground corn, ground oats, wheat middlings, alfalfa meal, corn gluten meal)

M.D. Basson et al. / Nutrition Research 24 (2004) 901 – 908

903

(Harlan Teklad, Madison, Wis). In addition, 5 experimental diets were prepared based on the AIN-93G diet. These were high fat (20% from soybean oil) without fiber, low fat (5% from soybean oil) without fiber, high fat with 10% (wt/wt) cellulose, high fat with 10% (wt/wt) wheat bran fiber, and high fat with 10% (wt/wt) guar gum fiber. The experimental diets were manufactured by Research Diets, Inc (New Brunswick, NJ). Table 1 contains some of the details on these diets. The Harlan Teklad 4.37% crude fiber diet and the weight by weight percentages of the high fat with 10% cellulose diet, high fat with 10% wheat bran diet, and the high fat with 10% guar gum diet corresponded to 18.2%, 11.1%, 9.8%, and 10.1% dietary fiber, respectively (analysis by Covance Laboratories, Madison, Wis). Further information (as provided by Research Diets, Inc) on the formulation of the diets is given in Table 1. The percent granulation of fibers used in diets on US Sieves for wheat bran and Mesh for cellulose were as follows: for wheat bran—on 14, 1%; on 20, 4%; on 40, 45%; on 80, 30%; through 80, 20%; and for cellulose—on 35, 0%; through 100, 100%; through 200, 85% to 95%. Table 1 Experimental diets Ingredient Casein, 80 Mesh l-Cystine Cornstarch Maltodextrin 10 Sucrose Cellulose Wheat bran Guar gum Soybean oil S10026c Dicalcium phosphate Calcium carbonate Potassium citrate Vitamin mixd Choline bitartrate Total a

HFa + NF b

200 3 120 100 220 0 0 0 200 10 13 5.5 16.5 10 2 900

LF + NF

HF + 10% C

HF + 10%W

HF + 10% G

200 3 289 100 388 0 0 0 50 10 13 5.5 16.5 10 2 1089

200 3 120 100 220 100 0 0 200 10 13 5.5 16.5 10 2 950

200 3 120 100 220 0 100 0 200 10 13 5.5 16.5 10 2 950

200 3 120 100 220 0 0 100 200 10 13 5.5 16.5 10 2 950

HF indicates 20% soybean oil; NF, no fiber; LF, 5% soybean oil; 10% C, 10% cellulose; 10% W, 10% wheat germ; and 10% G, 10% guar gum. b Values are expressed as weight (grams), unless noted otherwise. c s10026 salt mix consisting of the following components with the given percentage composition and with indicated concentrations in mix expressed as gram % (g%): sodium chloride, 39.3% Na, 60.7% Cl, 25.9; magnesium oxide, 60.3% Mg, 4.2; magnesium sulfated 7H2O, 9.87% Mg, 13.0% S, 25.8 g%; chromium potassium sulfated 12H2O, 10.4% Cr, 0.19 g%; cupric carbonate, 57.5% Cu, 0.11 g%; sodium fluoride, 45.2% Fl, 0.02 g%; potassium iodate, 59.3% I, 0.004 g%; ferric citrate, 21.2% Fe, 2.1 g%; manganous carbonate, 47.8% Mn, 1.23 g%; ammonium molybdated 4H2O, 54.3% Mo, 0.03 g%; sodium selenite, 45.7% Se, 0.004 g%; zinc carbonate, 52.1% Zn, 0.56 g%; sucrose, 39.9 g%. d Vitamin mix consisting of the following components with indicated concentrations in mix expressed as g%: vitamin A palmitate, 500,000 IU/g, 0.08 g%; vitamin D3, 100,000 IU/g, 0.10 g%; vitamin E acetate, 500 IU/g, 1.0 g%; menadione sodium bisulfite, 62.5% manadione, 0.008 g%; biotin, 1.0%, 0.2 g%; cyanococobalamin, 0.1%, 0.10 g%; folic acid, 0.02 g%; nicotinic acid, 0.3 g%; calcium pantothenate, 0.16 g%; pyridoxine HCl, 0.07 g%; riboflavin, 0.06 g%; thiamin HCl, 0.06 g%; sucrose, 97.8 g%.

904

M.D. Basson et al. / Nutrition Research 24 (2004) 901 – 908

2.3. Animal care Mice were randomly assigned to different diet groups and 4 mice receiving the same diet were caged in each cage. The mice received the diets for 30 days before sacrifice. Food consumption was monitored weekly in each cage of animals by weighing the food offered to each cage and then weighing the leftover each week. Animal body weight was measured immediately upon arrival and then weekly thereafter. Caloric intake was estimated as the product of food consumption and caloric density for each diet. Total fecal output of mice in each cage was weighed over a 24-hour period on the 30th day before sacrifice by CO2 inhalation. After sacrifice, the colons were immediately excised and formalin fixed for at least 24 hours. 2.4. Tissue processing and immunofluorescent staining Histochemical processing was performed in the Histopathology Unit of the Cancer Research UK, London, UK. Paraffin-embedded sections of the proximal colon were prepared from the formalin-fixed samples. The monoclonal antibody MIB-1 against the Ki-67 antigen, which is expressed in the nuclei of all cells in G1, S, and G2 phases and mitosis, but not in the G0 phase of the cell cycle, has become one of the most useful methods for assessment of cell growth in paraffin-embedded specimens of human tissue. Immunostaining was performed by using standard streptavidin-peroxidase technique. Briefly, sections were dewaxed and incubated in 100% alcohol and endogenous peroxidase (Nagarase, Cat No P-8038, Sigma, Shaftesbury, UK) activity was blocked by incubating in methanol containing 0.02% hydrogen peroxide for 10 minutes. Sections were then washed in running tap water, rinsed in phosphate-buffered saline (PBS) and incubated in swine serum in 1:25 dilution for 15 minutes. Sections were incubated for 35 minutes in primary antibody at a final dilution of 1:200 for 35 minutes (Triton Diagnostics, Flanders, NJ). Sections were then washed in PBS twice for 5 minutes each and incubated in biotinylated rabbit anti-mouse antibody (Dako E0353) at a final dilution of 1:500. Sections were again washed in PBS followed by incubation in streptavidin-peroxidase (Dako P 0397 Glostrup, Denmark) at 1:500 for 30 minutes. Sections were again washed in PBS for 10 minutes and incubated in peroxidase substrate, diaminobenzidine tetrahydrochloride (Sigma D-5637, Shaftesbury, UK), for 3 to 5 minutes. Peroxidase substrate was prepared as 5 mg diaminobenzidine tetrahydrochloride in 10 mL PBS, to which had been added 20 ll of 30% hydrogen peroxide. All sections were counterstained with Meyer’s hematoxylin, dehydrated in graded alcohols, and then mounted in DPX Mountant (Electron Microscopy Sciences, London, UK). Positive and negative (without primary antibody) controls were used. 2.5. Measurements of crypt length and proliferation Slides were examined and 30 intact crypts were identified. The crypt was then scored from the crypt base (position 0) to the end of the crypt. The slides were scored bblindQ in that the scorer was not aware of which group the tissues came from. The presence, number, and location of mitotic cells and labeled cells and the crypt length were recorded. The number of mitoses per crypt, the number of labeled cells per crypt, and the maximum and half-maximum labeling were determined. The growth fraction (the ratio of proliferating to nonproliferating cells) was taken as the point of half-maximum labeling expressed as a percentage of the crypt length.

M.D. Basson et al. / Nutrition Research 24 (2004) 901 – 908

905

2.6. Statistical analysis All results are presented as the mean F standard error of the mean. Data were analyzed by ANOVA to test the omnibus hypothesis and with Dunnett test to test multiple comparisons, with p b .05 being set a priori as the desired level of statistical significance. 3. Results All mice consumed the diets freely and gained weight similarly (not shown). To gauge the effect of consumption of each of these diets on cell proliferation, crypt length, expressed as the number of cells per crypt, mitoses per crypt, and Ki-67 labeling activity per crypt were measured. In addition, the percentage growth fraction was determined as the ratio of the halfmaximum labeling position to crypt length. Table 2 summarizes the effects of the different diets on the parameters of proliferation in mice consuming the different diets. As shown in Table 2, the length of the crypts were very similar, except for the cellulose group where the crypts were 18% shorter than the crypts of animals consuming the no-fiber, high-fat diet (p b .05). Turning our attention to the number of mitoses per crypt, we see that there was little difference between the animals consuming the Teklad diet and those consuming the no-fiber, high-fat diet. However, consumption of the no-fiber, low-fat diet was associated with a marked decrease in proliferation compared with the no-fiber, high-fat diet as measured by this parameter. Each of the defined-fiber, high-fat diets tended to be associated with a more modest decrease in proliferative activity compared with the no-fiber, high-fat dietary background. This did not achieve statistical significance for wheat bran or guar gum supplementation. However, the cellulose, high-fat diet was associated with a statistically significant decrease in mitosis per crypt compared with the no-fiber, high-fat diet (p b .05). Table 2 also displays the number of Ki-67–immunoreactive cells per crypt in mice consuming each diet. Results with this technique tended to be basically similar to those of the number of mitoses per crypt, although the number of MIB-1–labeled cells was much higher than the number of cells displaying mitotic figures. The no-fiber, high-fat diet was associated Table 2 Mean values of indicated measure of proliferation in mice fed the indicated diet Crypt length in cells Mitoses per crypt Ki-67–labeled cells per crypt Growth fraction (%) a b c

Teka

HF + NF

HF + 10% C

HF + 10% W

HF + 10% G

19.8b F 0.7

20.4 F 1.7

18.8 F 1.0

16.6 F 0.5

19.5 F 0.9

18.2 F 0.8

0.18 F 0.04

0.04c F 0.02

0.08 F 0.04

0.14 F 0.01

0.10 F 0.3

5.7c F 0.7

8.6 F 0.7

5.9c F 0.4

6.5c F 0.4

7.1 F 0.5

6.9 F 0.4

67.8 F 3.1

66.9 F 2.9

55.6 F 0.4

68.5 F 3.8

67.6 F 3.2

68.0 F 5.0

0.20 F 0.04

LF + NF

Tek indicates Harlan Teklad diet. Other abbreviations as in Table 1. Means F SEM. Entries with superscript c are statistically significant from control diet HF + NF ( p b .05).

906

M.D. Basson et al. / Nutrition Research 24 (2004) 901 – 908

with the greatest amount of proliferation as expressed by this parameter. The Teklad diet appeared to evoke the same amount of proliferation in the crypts as the no-fiber, low-fat diet and the defined-fiber, high-fat diets. In comparison with the no-fiber, high-fat diet, the cellulose, high-fat diet was the only defined-fiber, high-fat diet associated with a statistically significant difference in proliferation as expressed with the labeled cells per crypt parameter. There was a trend in mice consuming the other defined-fiber, high-fat diets toward decreased proliferation that did not achieve statistical significance. In addition, animals consuming the Teklad diet showed a slightly higher number of mitoses per crypt than the animals consuming the control diet. On the other hand, the difference in Ki-67–labeled cells per crypt was statistically significant between the animals consuming the Teklad diet and the control diet. Finally, Table 2 displays the growth fraction expressed as a percentage, derived from the ratio of the half-maximum labeling position to crypt length, also in cells. There were few differences among the diets, except that the no-fiber, low-fat diet was associated with a statistically significantly lower growth fraction than the no-fiber high-fat diet (p b .05).

4. Discussion The effect of lowered fat on mitotic rate was mirrored by an alteration in the growth fraction and labeling position within the crypt. This was not seen for approximately 10% dietary cellulose supplementation, but these results may have been confounded to some extent by a significant reduction in crypt length in the cellulose-fed animals. Microscopic observations of mitoses and Ki-67 labeling tended to yield parallel results, except for a disproportionally lower rate of Ki-67 labeling compared with rate of observed mitotic figures in the Teklad commercial diet group for unclear reasons. It is known, however, that intestinal proliferation indices will vary with nutritional, digesta, or systemic factors. Goodlad and Wright [9] found that superimposed on these factors are circadian and starvation/refeeding factors that also affect proliferation. The Teklad diet or its digesta may have uncharacterized mitogenic or antimitogenic factors not present in the other defined diets in this study. In addition, the time of sacrifice of these animals was not synchronized to a feeding schedule because the animals were fed ad libitum. Hence, the discordant results between the number of mitoses per crypt and the Ki-67 labeling per crypt may not be wholly unexpected. The observation that colonocyte proliferation was proportional to soybean oil fat intake is certainly consistent with previous observations by others of fat stimulation of colonic proliferation in various diets and models [10], although at least one investigator failed to document stimulation of proliferation by fat [11]. The mechanism for this effect is unclear, but has been variously hypothesized to reflect cytotoxicity [12,13], the modulation of intracellular signals such as PKC [14,15], or interactions with other dietary constituents [13,16,17]. In the present study, no increase in mitoses per crypt or labeling per crypt was observed with the supplementation of any of the fibers studied on a background of a 20% soybean oil diet. Indeed, a tendency was even observed for some inhibition with supplementation with wheat bran or guar gum, although this was not statistically significant. Furthermore, cellulose

M.D. Basson et al. / Nutrition Research 24 (2004) 901 – 908

907

supplementation actually appeared to inhibit cell proliferation. The mechanism for this effect is not clear. Review of the literature illustrates profound differences in the results of fiber on the bowel depending on the fiber itself, the diet it is added to, and even the preparation of the fiber [18]. Likewise, reports in human subjects on the effects of dietary fiber on risk of developing colonic lesions and recurrences of adenomas are contradictory. Some recent reports point to no effect on risk of developing colonic lesions [19] and recurrences of adenomas [20], whereas other recent reports show a reduction in risk of development of colonic cancer [21] and recurrences of adenomas [22]. However, we do note that cellulose supplementation results in substantially bulkier stools in these mice, presumably because of its poorly fermentable hygroscopic nature [1]. Stool bulk was not measured in several of the previous fiber studies reported. However, in this study, stool bulk was most markedly increased by cellulose supplementation. Because fiber supplementation that increases stool bulk and decreases stool hardness has been demonstrated to reduce pressure within the colonic lumen [8,23], it is possible that such a reduction might have reduced force-stimulated mucosal signaling and proliferation [6,24-26], contributing to the observed decrease in colonic mucosal proliferation, although this hypothesis awaits further study and clarification. It is certainly alternatively possible that fiber could have acted to dilute or bind mitogenic agents associated with the soybean oil supplement. Our data show a trend toward decreased colonic proliferation in mice fed the high-fat plus fiber diets in comparison to the high-fat plus no-fiber control diet. In the specific case of cellulose, the difference reached statistical significance for the Ki-67 labeling position. A decrease in proliferation mediated via a reduction in intraluminal strain and/or pressure as an explanation is an attractive hypothesis. It may be that cellulose produced a more profound decrease in this strain and/or pressure when compared with the other fiber types used in this study. Another possibility is that this study lacks the statistical power to reveal small differences in effects among the different types of fiber-supplemented diets. The conflicting results among reports on the effect of fiber and fat on colonic proliferation in various human and animal studies suggest the complexity of the regulation of colonic mucosal proliferation in different settings and the multitude of effects these dietary elements may exert. Our results reinforce the need for further study to elucidate this important issue. References [1] Yu CF, Whiteley L, Carryl O, Basson MD. Differential dietary effects on colonic and small bowel neoplasia in C57BL/6J Apc Min/+ mice. Dig Dis Sci 2001;46:1367 - 80. [2] Basson MD, Li GD, Hong F, Han O, Sumpio BE. Amplitude-dependent modulation of brush border enzymes and proliferation by cyclic strain in human intestinal Caco-2 monolayers. J Cell Physiol 1996;168:476 - 88. [3] Murnin M, Kumar A, Li GD, Brown M, Sumpio BE, Basson MD. Effects of glutamine isomers on human (Caco-2) intestinal epithelial proliferation, strain-responsiveness, and differentiation. J Gastrointest Surg 2000;4:435 - 42. [4] Han O, Li GD, Sumpio BE, Basson MD. Strain induces Caco-2 intestinal epithelial proliferation and differentiation via PKC and tyrosine kinase signals. Am J Physiol 1998;275:G534 - 41. [5] Li W, Duzgun A, Sumpio BE, Basson MD. Integrin and FAK-mediated MAPK activation is required for cyclic strain mitogenic effects in Caco-2 cells. Am J Physiol Gastrointest Liver Physiol 2001;280:G75 - G87.

908

M.D. Basson et al. / Nutrition Research 24 (2004) 901 – 908

[6] Zhang J, Li W, Sanders MA, Sumpio BE, Panja A, Basson MD. Regulation of the intestinal epithelial response to cyclic strain by extracellular matrix proteins. FASEB J 2003;17:926 - 8. [7] Basson MD, Coppola CP. Repetitive deformation and pressure activate small bowel and colonic mucosal tyrosine kinase activity in vivo. Metab Clin Exp 2002;51:1525 - 7. [8] Murakami H, Iwane S, Munakata A, Nakaji S, Sugawara K, Tsuchida S, Sasaki D. Changes in intraluminal pressure in rat large intestines with aging and effects of dietary fiber. Dig Dis Sci 2001;46:1247 - 54. [9] Goodlad RA, Wright NA. The effect of starvation and refeeding on intestinal cell proliferation in the mouse. Virchows Arch B Pathol 1984;45:63 - 73. [10] Reddy BS. Dietary fat and colon cancer: animal model studies. Lipids 1992;27:807 - 13. [11] Sesink AL, Termont DS, Kleibeuker JH, Van Der Meer R. Red meat and colon cancer: dietary haem, but not fat, has cytotoxic and hyperproliferative effects on rat colonic epithelium. Carcinogenesis 2000;21:1909 - 15. [12] Bird RP, Medline A, Furrer R, Bruce WR. Toxicity of orally administered fat to the colonic epithelium of mice. Carcinogenesis 1985;6:1063 - 6. [13] Stadler J, Stern HS, Yeung KS, McGuire V, Furrer R, Marcon N, et al. Effect of high fat consumption on cell proliferation activity of colorectal mucosa and on soluble faecal bile acids. Gut 1988;29:1326 - 31. [14] Chapkin RS, Gao J, Lee DY, Lupton JR. Dietary fibers and fats alter rat colon protein kinase C activity: correlation to cell proliferation. J Nutr 1993;123:649 - 55. [15] Pajari AM, Oikarinen S, Grasten S, Mutanen M. Diets enriched with cereal brans or inulin modulate protein kinase C activity and isozyme expression in rat colonic mucosa. Br J Nutr 2000;84:635 - 43. [16] Pell JD, Gee JM, Wortley GM, Johnson IT. Dietary corn oil and guar gum stimulate intestinal crypt cell proliferation in rats by independent but potentially synergistic mechanisms. J Nutr 1992;122:2447 - 56. [17] Lee DY, Chapkin RS, Lupton JR. Dietary fat and fiber modulate colonic cell proliferation in an interactive site-specific manner. Nutr Cancer 1993;20:107 - 18. [18] Brunsgaard G. Effects of cereal type and feed particle size on morphological characteristics, epithelial cell proliferation, and lectin binding patterns in the large intestine of pigs. J Anim Sci 1998;76:2787 - 98. [19] Fuchs CS, Giovannucci EL, Colditz GA, Hunter DJ, Stampfer MJ, Rosner B, Speizer FE, Willett WC. Dietary fiber and the risk of colorectal cancer and adenoma in women. N Engl J Med 1999;340:169 - 76. [20] Schatzkin A, Lanza E, Corle D, Lance P, Iber F, Caan B, Shike M, Weissfeld J, Burt R, Cooper MR, Kikendall JW, Cahill J. Lack of effect of a low-fat, high-fiber diet on the recurrence of colorectal adenomas. Polyp Prevention Trial Study Group. N Engl J Med 2000;342:1149 - 55. [21] Bingham SA, Day NE, Luben R, Ferrari P, Slimani N, Norat T, Clavel-Chapelon F, Kesse E, Nieters A, Boeing H, Tjonneland A, Overvad K, Martinez C, Dorronsoro M, Gonzalez CA, Key TJ, Trichopoulou A, Naska A, Vineis P, Tumino R, Krogh V, Bueno-de-Mesquita HB, Peeters PH, Berglund G, Hallmans G, Lund E, Skeie G, Kaaks R, Riboli E. Dietary fibre in food and protection against colorectal cancer in European Prospective Investigation into Cancer and Nutrition (EPIC): an observational study. Lancet 2003;361:1496 - 501. [22] Peters U, Sinha R, Chatterjee N, Subar AF, Ziegler RG, Kulldorff M, Bresalier R, Weissfeld JL, Flood A, Schatzkin A, Hayes RB; Prostate, Lung, Colorectal, and Ovarian Cancer Screening Trial Project Team. Dietary fibre and colorectal adenoma in a colorectal cancer early detection programme. Lancet 2003;361: 1491 - 5. [23] Brodribb AJ, Condon RE, Cowles V, DeCosse JJ. Effect of dietary fiber on intraluminal pressure and myoelectric activity of left colon in monkeys. Gastroenterology 1979;77:70 - 4. [24] Basson MD, Coppola C. Repetitive deformation and pressure activate small bowel and colonic mucosal tyrosine kinase activity in vivo. Metabolism 2002;51:1525 - 7. [25] Basson MD. Paradigms for mechanical signal transduction in the intestinal epithelium. Digestion 2003;68:217 - 25. [26] Thamilselvan VJ, Basson MD. Pressure activates colon cancer cell adhesion by inside-out focal adhesion complex and actin cytoskeletal signaling. Gastroenterology 2004;126:8 - 18.