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Effects of high-amylose maize starch and butyrylated high-amylose maize starch on azoxymethane-induced intestinal cancer in rats

Effects of high-amylose maize starch and butyrylated high-amylose maize starch on... Carcinogenesis vol.29 no.11 pp.2190–2194, 2008 doi:10.1093/carcin/bgn192 Advance Access publication August 13, 2008 Effects of high-amylose maize starch and butyrylated high-amylose maize starch on azoxymethane-induced intestinal cancer in rats 1,2, 1,2 1,2 of short chain fatty acids (SCFA). Consumption of RS by humans has Julie M.Clarke , David L.Topping , Anthony R.Bird , 1,3 4 been shown to improve the colonic environment by softening stools, Graeme P.Young and Lynne Cobiac increasing faecal bulk, decreasing faecal pH, increasing luminal SCFA 1 2 Preventative Health National Research Flagship and CSIRO Human Nutrition, concentrations (3) and reducing the accumulation of potentially harm- Adelaide, South Australia, Flinders Cancer Control Alliance, Flinders ful by-products of protein fermentation (4). Recent animal studies show University, Bedford Park, South Australia and Department of Nutrition and that RS prevents colonic DNA damage in rats fed high protein diets (5). Dietetics, School of Medicine, Flinders University, Bedford Park, South The main SCFA produced by colonic fermentation of carbohydrate Australia are acetate, propionate and butyrate. These SCFA have important roles To whom correspondence should be addressed. CSIRO Human Nutrition, PO in the maintenance of bowel health that include increasing colonic blood Box 10041, Adelaide BC 5000, South Australia. Tel: þ61 8 8303 8800; flow, improving mineral and water absorption from the colon and the Fax: þ61 8 8303 8899. maintenance of low colonic pH (6). Butyrate, which is the preferred Email: [email protected] energy source for colonocytes (7), may beprotectiveagainst largebowel Colorectal cancer (CRC) is a major cause of death worldwide. cancer by inhibiting histone deacetylase thereby enhancing the apoptotic Studies suggest that dietary fibre offers protection perhaps by deletion of genetically damaged cells (8) through a histone hyperacety- increasing colonic fermentative production of butyrate. This lation-mediated pathway (9,10). Butyrate has also been shown to inhibit study examined the importance of butyrate by investigating the proliferation and induce differentiation of cancer cells in vitro (11,12). effects of resistant starch (RS) and butyrylated-RS on azoxyme- Strategies to increase colonic levels of SCFA, particularly butyrate, thane (AOM)-induced CRC in rats. Four groups (n 5 30 per may have clinical and public health benefits. However, there may be group) of Sprague–Dawley rats were fed AIN-93G-based diets limits to the usefulness of promoting RS consumption to increase containing a standard low-RS maize starch (LAMS), LAMS 1 butyrate levels as the microflora of some individuals have limited 3% tributyrin (LAMST), 10% high-amylose maize starch capacity to ferment certain types of RS, the SCFA profile produced (HAMS) and 10% butyrylated HAMS (HAMSB) for 4 weeks. can vary with RS source (13) and dietary fibre is not universally Rats were injected once weekly for 2 weeks with 15 mg/kg tolerated. Starches acylated to a degree of substitution of 0.2–0.25 AOM, maintained on diets for 25 weeks and then killed. Butyrate (i.e. 0.2–0.25 of the hydroxyl groups on each starch D-glucopyranosyl concentrations in large bowel digesta were higher in rats fed unit were derivatized or replaced by substituent acids) have been HAMSB than other groups (P < 0.001); levels were similar in shown to be an effective means of delivering specific SCFA to the HAMS, LAMS and LAMST groups. The proportion of rats de- colon in both animals (14,15) and humans (16). The SCFA, esterified veloping tumours were lower in HAMS and HAMSB than LAMS to the starch carrier molecule, are liberated by bacterial enzymes and (P < 0.05), and the number of tumours per rat were lower in are absorbed and utilized by the colonocytes or gut microbes. HAMSB than LAMS (P < 0.05). Caecal digesta butyrate pools This study investigated the importance of butyrate by examining and concentrations were negatively correlated with tumour size the effects of RS and butyrylated-RS on tumour development in rats (P < 0.05). Hepatic portal plasma butyrate concentrations were treated with azoxymethane (AOM). The effect of systemic butyrate higher (P < 0.001) in the HAMSB compared with other groups absorbed from the small intestine was also examined by the inclusion and negatively correlated with tumour number per rat (P < 0.009) of rats fed tributyrin (LAMST). and total tumour size for each rat (P 5 0.05). HAMSB results in higher luminal butyrate than RS alone or tributyrin. This is asso- Materials and methods ciated with reduced tumour incidence, number and size in this rat model of CRC supporting the important protective role of Animals and diets butyrate. Interventional strategies designed to maximize luminal Adult male Sprague–Dawley rats (198 ± 2 g) were purchased from the Animal butyrate may be of protective benefit in humans. Resource Centre, Murdoch University, Western Australia. The rats were housed in wire-bottomed cages in a temperature-controlled room (22–24C) with a 12 h light–dark cycle (lights on 8:00–20:00). They were allocated randomly into four groups (n 5 30 per group) with approximately equal body weights and given free access to water and diet throughout the study. Introduction The experimental diets were based on a control AIN-93G (17) diet that Colorectal cancer (CRC) is a common malignancy in Western socie- contained 530 g standard low-amylose maize starch, 200 g casein, 70 g corn oil and 50 g a-cellulose/kg of diet. The choline, methionine, minerals, vitamins ties resulting in 8% of all cancer deaths (1) and is emerging as a sig- and antioxidant were added as described previously (15). The amount of stan- nificant cause of morbidity and mortality in several Asian and Eastern dard maize starch in the diet was reduced to allow for the addition of exper- European countries as they adopt western dietary habits and lifestyles. imental starches and tributyrin. The experimental diets were control (LAMS, This cancer is considered mostly preventable by appropriate diet and low RS), low-amylose maize starch with 3% tributyrin (LAMST, low RS), 10% associated lifestyle factors (2). high-amylose maize starch (HAMS, high RS) or 10% butyrylated HAMS Resistant starch (RS) is dietary starch that escapes digestion in the (HAMSB, high RS). The latter was manufactured by National Starch Food small intestine. It is fermented by colonic bacteria with the production Innovation and had a degree of substitution of 0.25. HAMS and HAMSB were cooked with water and spray dried (to minimize crystallite formation) as de- scribed previously (18) before addition to the diets. This process was used to Abbreviations: AOM, azoxymethane; CRC, colorectal cancer; CSIRO, Com- mimic the condition of starches consumed in foods by humans as cooking monwealth Scientific and Industrial Research Organisation; HAMS, high- changes the production and delivery of butyrate to the colon by starches amylose maize starch; HAMSB, butyrylated high-amylose maize starch; LAMS, (15). All diets were prepared regularly at CSIRO and stored at 4C. The rats low-amylose maize starch; RS, resistant starch; SCFA, short chain fatty acids. were fed fresh diet daily. The Author 2008. Published by Oxford University Press. All rights reserved. For Permissions, please email: [email protected] The online version of this article has been published under an open access model. Users are entitled to use, reproduce, disseminate, or display the open access version of this article for non-commercial purposes provided that: the original authorship is properly and fully attributed; the Journal and Oxford University Press are attributed as the original place of publication with the correct citation details given; if an article is subsequently reproduced or disseminated not in its entirety but only in part or as a derivative work this must be clearly indicated. For commercial re-use, please contact [email protected] Resistant starch and butyrate on colon cancer in the rat After 28 days of feeding the experimental diets rats were injected with Caecal tissue and digesta weights were higher in rats fed HAMSB 15 mg of AOM/kg (Sigma Chemical Co., St Louis, MO). The rats were main- than all other groups (P , 0.001), and caecal tissue weight was high- tained on their experimental diets and were injected with the same dose of er in HAMS rats compared with LAMST (P , 0.01) (Table I). Caecal AOM a week later. They were monitored closely until the termination of the digesta pH was different between groups with pH in HAMSB-fed rats experiment 25–27 weeks post-initial AOM injection. Rats were anaesthetized lower than the other groups (P , 0.001), and the pH in the HAMS with halothane and blood collected into heparin-treated tubes from the hepatic group lower than the LAMS (P , 0.001) and LAMST (P , 0.05) portal vein. The small and large intestines and caecum were opened longitu- groups (Table I). The digesta pH fell from caecum to distal colon in all dinally, digesta collected and the epithelial surface cleaned with 0.15 mM groups except HAMSB; in the distal colon, the digesta pH of rats fed NaCl/l. The colon was divided into two equal length parts designated proximal LAMS was higher than HAMS (P , 0.01). and distal colon. The location, number and size of visible tumours were re- corded for each animal before the tumours were collected and fixed in 10% Caecal and proximal and distal colonic digesta butyrate concentra- buffered formalin, embedded in paraffin blocks and processed by routine his- tions were higher in rats fed HAMSB than other groups (P , 0.001) tological procedures for subsequent haematoxylin and eosin staining. and were similar in HAMS, LAMS and LAMST groups (Figure 1). All procedures involving animals were approved by the CSIRO Human Digesta SCFA pool sizes, calculated by multiplying the digesta wet Nutrition Animal Ethics Committee and complied with the Australian Code weights by the concentrations of each of the fatty acids measured of Practice (19). (acetate, propionate and butyrate), are shown in Table II. Digesta butyrate pool sizes were consistently larger throughout the large Analytical procedures bowel in rats fed HAMSB (P , 0.05–0.001). Caecal pools of acetate Analysis of SCFA, dry matter and pH was undertaken as described previously and propionate and proximate and distal colonic pools of propionate (15,20). All tumours were examined histologically by a single independent observer unaware of the dietary treatment and categorized into adenomas and were also larger in rats fed HAMSB (P , 0.001–0.05). Caecal and adenocarcinomas (invasive tumours) based on the criteria described previously distal pools of propionate were higher in HAMS than LAMST or (21). The ‘tumour incidence’ was defined as the proportion of rats in a group LAMS groups (P , 0.05–0.001), and proximal pools of propionate that had at least one tumour in the small or large bowel. The ‘size’ of each were higher in HAMS than LAMST (P , 0.001). tumour was calculated using the formula: Hepatic portal plasma butyrate concentration was higher (P , 0.001) in the HAMSB compared with the other groups (Figure 1). Dietary Tumour area index5p½ðD1  D2Þ=4 tributyrin (LAMST) increased hepatic portal butyrate concentration compared with LAMS to levels similar to HAMS (P , 0.001). where D and D are the length and width of each tumour. 1 2 More large bowel tumours were found in the distal colon rather Statistical analyses than the proximal colon (P , 0.02); however, a greater proportion of GraphPad Prism version 4.0 for Windows (http://www.graphpad.com) com- proximal tumours was adenocarcinomas (13 of 32) compared with the puter software was used for statistical analyses. Parametric data (body weight, distal colon, where most tumours were adenomas (55 of 67) organ weights and digesta measures) were analysed using one-way analysis of (P , 0.02). The size of caeco-colonic adenomas was positively and variance and Tukey’s post hoc multiple comparison test. Tumour indices were significantly correlated to the distance of the adenomas from the rectum compared between treatment groups using either Kruskal–Wallis test with (Spearman r 5 0.310, P , 0.008, n 5 74) with larger adenomas tend- Dunn’s post hoc tests or Mann–Whitney two-tailed t-tests for non-parametric ing to be closer to the caecum. The reverse was the case for adenocar- data. Tumour incidence data were analysed using Pearson’s v test using cinomas, with larger adenocarcinomas tending to be closer to the contingency tables. The relationships between butyrate and tumour measures rectum (Spearman r 5 0.403, P , 0.05, n 5 25). There was no were determined by non-parametric Spearman-ranked correlations. Data effect of diet on the distribution of tumours based on size or tumour are expressed as mean ± SEM with statistical significance indicated when P , 0.05. type i.e. the ratio of adenomas to adenocarcinomas in the proximal and distal colons was not significantly different between treatment groups. Large bowel tumour incidence (proportion of rats per group with Results tumours) was lower in HAMSB and HAMS compared with LAMS group (P , 0.05). Tumour number (number of tumours per group) Growth rates were similar in all the experimental groups and there was lower in HAMSB compared with LAMS (P , 0.05). There was were no significant differences in final body weights between the dietary treatments (mean for all groups 627 ± 7 g, Table I). Two rats were euthanased early due to events unrelated to the experimental procedures (from group LAMS in week 9; HAMSB, week 23). Seven rats were euthanased within 4 weeks of their scheduled dates because they showed possible symptoms of CRC including weight loss and rectal bleeding; tumour end points were obtained from these animals. Table I. Final measures of rats fed diets containing low-amylose maize starch (LAMS), LAMS with 3% tributyrin (LAMST), HAMS and butyrylated HAMS (HAMSB) LAMS LAMST HAMS HAMSB Body weight (g) 635 ± 15 621 ± 18 638 ± 14 616 ± 25 a a,b a Caecal tissue 0.84 ± 0.02 0.78 ± 0.02 0.93 ± 0.03 1.19 ± 0.03 weight (g) a a a Caecal digesta (g) 1.46 ± 0.07 1.41 ± 0.09 1.70 ± 0.09 2.27 ± 0.08 a,c a a ,d Caecal digesta pH 7.12 ± 0.04 7.07 ± 0.04 6.92 ± 0.03 6.69 ± 0.04 Distal colonic 6.97 ± 0.05 6.85 ± 0.07 6.66 ± 0.05 6.77 ± 0.06 Fig. 1. Concentration of butyrate in the digesta of the caecum, proximal and digesta pH distal colon (mM; n 5 28–30) and hepatic portal plasma (mM; n 5 26–30) of rats fed diets containing low-amylose maize starch (LAMS; open bars), Values are mean ± SEM, n 5 28–30. LAMS with 3% tributyrin (LAMST; light grey bars), (HAMS; dark grey Significantly different from HAMSB, P , 0.001. bars) and butyrylated HAMS (HAMSB; black bars) (mean ± SEM). b a b Significantly different from HAMS, P , 0.01. Significantly different from HAMSB, P , 0.001; significantly different c c Significantly different from HAMS, P , 0.001. from HAMS, P , 0.05 and significantly different from LAMST, P , 0.001 Significantly different from HAMS, P , 0.005 within each row of data. within each gut region or hepatic portal plasma. 2191 J.M.Clarke et al. an apparent trend towards reduced large bowel number for the HAMS particularly butyrate, to the colon (22). Low levels of colonic SCFA group compared with the LAMS (P , 0.055). Caecal butyrate pools in individual humans may result either from low consumption of di- and concentrations were significantly and negatively correlated with etary fibre or from an inadequate fermentative capacity of the colonic the number of large bowel tumours per rat (P , 0.03, r 5 0.206 and microflora. Earlier animal studies have shown that acylated starches P , 0.04, r 50.198, respectively), whereas there was no relation- are twice as effective as HAMS in raising large bowel SCFA levels of ship between distal butyrate levels and tumour number. The total the acid that has been esterified (14,15). The present data confirm tumour area for each rat was lower in the HAMBS than LAMS group those previous experiments and show that while both HAMS and (P , 0.05). The caecal butyrate pools and total large bowel tumour HAMSB raise total SCFA, the greatest increase in butyrate was with area for each rat were significantly and negatively correlated the butyrylated SCFA. HAMSB was particularly effective in raising (P , 0.05, r 5 0.185). Hepatic portal plasma butyrate concentra- butyrate concentrations in the distal colon, which is the site of most tion was negatively correlated with tumour number per rat (Spearman CRC in humans. r 5 0.243, P , 0.009) and total tumour area index for each rat This study examined the importance of butyrate by investigating (Spearman r 5 0.184, P 5 0.05). the effects of RS (a dietary fibre) and butyrylated-RS on levels of Most small intestinal adenomas were found in the proximal small butyrate in the large bowel and their impact on intestinal tumorigen- intestine (data not shown). There were no significant differences in the esis in an established animal model of CRC. The results from this incidence, numbers and ratio of adenomas to adenocarcinomas in the study are consistent with previous experiments showing that dietary small intestine between groups (Table III). Tumours in the small in- RS reduces CRC incidence in rats treated with AOM (23–26). The testine of rats fed LAMST were significantly larger than those in the importance of butyrate to this protection is demonstrated by the ability rats fed LAMS (P , 0.05) but the total small intestinal tumour area of an RS-equivalent HAMSB to achieve greater protection and higher per rat was not significantly affected by diet. butyrate levels. Statistical examination of the data showed that this effect correlated with caecal butyrate pools and concentrations and hepatic portal butyrate concentrations. Discussion The total tumour size per rat was also significantly lower in the Acylated starches were designed to promote large bowel health by HAMSB than LAMS group, which reflects a lower incidence and providing a vehicle for the rapid and sustained delivery of SCFA, smaller size of tumours in rats fed HAMSB. This further supports Table II. Digesta SCFA pools (micromoles) of rats fed diets containing low-amylose maize starch (LAMS), LAMS with 3% tributyrin (LAMST), HAMS and butyrylated HAMS (HAMSB) Diet Caecal SCFA pools Proximal colon SCFA pools Distal colon SCFA pools Acetate Propionate Butyrate Acetate Propionate Butyrate Acetate Propionate Butyrate a a,b a a a a,c a LAMS 107.7 ± 7.2 30.0 ± 1.8 21.3 ± 1.6 44.1 ± 4.2 11.4 ± 1.1 7.1 ± 0.7 40.9 ± 3.8 9.5 ± 0.8 7.8 ± 0.8 a a,b a b a,c a c a,c a LAMST 99.8 ± 7.6 27.8 ± 2.3 22.0 ± 1.7 31.6 ± 3.1 8.1 ± 0.7 5.3 ± 0.6 36.7 ± 4.1 8.7 ± 0.9 7.6 ± 1.0 a a a a a d d HAMS 132.2 ± 11.8 53.5 ± 4.6 36.2 ± 4.0 47.2 ± 4.8 17.2 ± 1.6 9.2 ± 0.9 55.4 ± 5.7 20.8 ± 2.1 12.8 ± 1.9 HAMSB 237.2 ± 26.8 125.7 ± 11.1 125.5 ± 12.5 45.5 ± 4.3 23.5 ± 2.3 18.7 ± 2.0 51.1 ± 5.0 29.5 ± 2.9 19.4 ± 2.0 Values are mean ± SEM, n 5 28-30 for caecal; 24–30 for proximal colon; 24–27 for distal colon. Significantly different from HAMSB, P , 0.001. Significantly different from HAMS, P , 0.05. Significantly different from HAMS, P , 0.001. Significantly different from HAMSB, P , 0.05 within each column of data. Table III. Indices of AOM-induced small and large bowel tumours in rats fed diets containing low-amylose maize starch (LAMS), LAMS with 3% tributyrin (LAMST), HAMS and butyrylated HAMS (HAMSB) (mean ± SEM) LAMS LAMST HAMS HAMSB Number of rats 29 30 30 29 Incidence of tumours (%) Small intestine 27.6 16.7 30.0 20.7 b c,d Large bowel 65.5 56.7 40 37.9 Number tumours per rat Small intestine 0.4 0.2 0.4 0.2 e c Large bowel 1.3 0.9 0.6 0.5 Average tumour area (tumour area index) Small bowel 35.9 ± 11.5 146.9 ± 70.5 49.0 ± 10.2 51.8 ± 11.0 Large bowel 26.2 ± 8.8 21.9 ± 4.6 49.5 ± 14.7 18.0 ± 5.1 Total tumour area per rat Small bowel 13.6 ± 5.8 29.4 ± 17.3 19.6 ± 6.9 10.7 ± 4.5 e c Large bowel 34.3 ± 12.7 19.8 ± 4.7 31.4 ± 12.7 9.3 ± 3.5 Tumour type (large intestine) Adenoma (number) 27 24 12 11 Adenocarcinoma (number) 11 3 7 4 a 2 Analysed using Pearson’s v test. Contingency comparison between two groups. Significantly different from HAMSB, P , 0.05. Significantly different from HAMS, P , 0.05. Analysed using Mann–Whitney non-parametric t-tests between two groups. Significantly different from LAMST, P , 0.05. 2192 Resistant starch and butyrate on colon cancer in the rat the argument that butyrate absorbed from the lumen of the large bowel cose are preferred ahead of SCFA as energy substrates by jejunal may have a role regulating the initiation and growth of large bowel enterocytes (38) that would normally be exposed to low-digesta tumours. butyrate levels, suggesting nutritional factors controlling cellular The relationship between tumour size and butyrate pool was stron- apoptosis may differ in the small and large intestine. Other workers ger for the caecum than the colon, which may reflect the large bowel have reported an increase in small intestinal tumour incidence, but physiology of the rat in which bacterial activity occurs principally in not size, in response to RS (39) in mice with a targeted mutant gene the caecum. resulting in spontaneous small intestinal tumours. In the AOM- There was no effect of tributyrin on tumour development in the treated rats in this study, HAMS did not increase small intestinal large bowel. Tributyrin (glycerol tributyrate) is absorbed in the small tumour size or incidence, supporting the epidemiological evidence intestine, raising free butyrate concentrations in peripheral plasma for that high-RS diets do not enhance the risk of tumourigenesis in the up to 4 h (27). Tributyrin has been recommended for clinical evalu- small intestine (40). ation as a possible treatment for leukaemia (28) but has not been The current study demonstrates that large bowel and hepatic portal found to reduce the incidence of AOM-induced CRC in mice (29). venous butyrate levels correlated negatively with tumour indices in an At the level of tributyrin included in the LAMST diet in this study accepted animal model of CRC. Butyrate was raised by HAMS rela- (3%), hepatic portal plasma butyrate concentrations were similar to tive to the LAMS control, but the highest butyrate levels were those that resulted from ingesting the HAMS diet. In a previous study achieved with HAMSB, indicating the greater potential effectiveness (unpublished data), hepatic portal butyrate concentrations up to 1.0 for this modified starch to significantly improve bowel health and mM were recorded in individual rats fed 3% tributyrin; however, these reduce the risk of developing CRC. Further work is necessary to de- levels were not associated with increased butyrate concentrations in termine the effectiveness of HAMSB in opposing initiation or pro- peripheral plasma. These data suggest that 3% tributyrin may not gression of experimental carcinogenesis. increase levels of systemically delivered butyrate to the large bowel and that butyrate absorbed from the small intestine in the rat is in- Acknowledgements effective in altering large bowel CRC induced by AOM. The authors wish to thank National Starch Food Innovation for providing Genomic instability is a prerequisite for oncogenesis and the gen- the HAMS and HAMSB and gratefully acknowledge Julie Dallimore, otoxic effects of AOM are thought to be responsible for tumour Ben Scherer, Jessica Winkler and Peter Royle for their contributions to the initiation leading to the development of aberrant crypts, adenomas study. In addition, the statistical advice of Dr Ian Saunders of CSIRO Math- and eventually invasive adenocarcinoma. Butyrate could inhibit this ematical and Information Sciences (Urrbrae, South Australia) was greatly process either by promoting apoptosis (8) or repair in damaged appreciated. colonocytes during the initiation phase or by suppressing the growth of cells with damaged DNA (30). Alternatively, butyrate may be Conflict of Interest Statement: None declared. reducing tumour growth by inhibiting hypoxia-induced angiogenesis (31,32). It is not clear which mechanism(s) is operating. Tumour numbers and incidence were lower in HAMSB than in LAMS. References The size of neoplastic lesions is an important factor clinically as 1. Garcia,M. et al. Global Cancer Facts & Figures 2007. American Cancer large adenomas are more likely to progress to adenocarcinomas Society. Atlanta, GA. (33). Progression to adenocarcinoma may be the consequence of 2. World Cancer Research Fund. 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(2006) Dietary resistant starch type 3 prevents colonic DNA double-strand breaks than white meat in rats: attenuation tumor induction by 1,2-dimethylhydrazine and alters proliferation, apopto- by high amylose maize starch. Carcinogenesis, 28, 2355–2362. sis and dedifferentiation in rat colon. Carcinogenesis, 27, 1849–1859. 37. Potten,C.S. (1997) Epithelial cell growth and differentiation. 2. Intestinal 24. Le Leu,R.K. et al. (2007) Effect of dietary resistant starch and protein on apoptosis. Am. J. Physiol., 273, G253–G257. colonic fermentation and intestinal tumourigenesis in rats. Carcinogenesis, 38. Fleming,S.E. et al. (1991) Nutrient utilization by cells isolated from rat 28, 240–245. jejunum, cecum and colon. J. Nutr., 121, 869–878. 25. Jacobsen,H. et al. (2006) Carbohydrate digestibility predicts colon carci- 39. Williamson,S.L.H. et al. (1999) Intestinal tumorigenesis in the Apc1638N nogenesis in azoxymethane-treated rats. Nutr. Cancer, 55, 163–170. mouse treated with aspirin and resistant starch for up to 5 months. 26. Le Leu,R.K. et al. (2007) Suppression of azoxymethane-induced colon Carcinogenesis, 20, 805–810. cancer development in rats by dietary resistant starch. Cancer Biol. Ther., 40. Cassidy,A. et al. (1994) Starch intake and colorectal cancer risk: an 6, 1621–1626. international comparison. Br. J. Cancer, 69, 937–942. 27. Egorin,M.J. et al. (1999) Plasma pharmacokinetics of butyrate after intra- venous administration of sodium butyrate or oral administration of tribu- Received March 19, 2008; revised July 13, 2008; accepted August 1, 2008 http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Carcinogenesis Pubmed Central

Effects of high-amylose maize starch and butyrylated high-amylose maize starch on azoxymethane-induced intestinal cancer in rats

Clarke, Julie M.; Topping, David L.; Bird, Anthony R.; Young, Graeme P.; Cobiac, Lynne
Carcinogenesis , Volume 29 (11) – Aug 13, 2008
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Carcinogenesis vol.29 no.11 pp.2190–2194, 2008 doi:10.1093/carcin/bgn192 Advance Access publication August 13, 2008 Effects of high-amylose maize starch and butyrylated high-amylose maize starch on azoxymethane-induced intestinal cancer in rats 1,2, 1,2 1,2 of short chain fatty acids (SCFA). Consumption of RS by humans has Julie M.Clarke , David L.Topping , Anthony R.Bird , 1,3 4 been shown to improve the colonic environment by softening stools, Graeme P.Young and Lynne Cobiac increasing faecal bulk, decreasing faecal pH, increasing luminal SCFA 1 2 Preventative Health National Research Flagship and CSIRO Human Nutrition, concentrations (3) and reducing the accumulation of potentially harm- Adelaide, South Australia, Flinders Cancer Control Alliance, Flinders ful by-products of protein fermentation (4). Recent animal studies show University, Bedford Park, South Australia and Department of Nutrition and that RS prevents colonic DNA damage in rats fed high protein diets (5). Dietetics, School of Medicine, Flinders University, Bedford Park, South The main SCFA produced by colonic fermentation of carbohydrate Australia are acetate, propionate and butyrate. These SCFA have important roles To whom correspondence should be addressed. CSIRO Human Nutrition, PO in the maintenance of bowel health that include increasing colonic blood Box 10041, Adelaide BC 5000, South Australia. Tel: þ61 8 8303 8800; flow, improving mineral and water absorption from the colon and the Fax: þ61 8 8303 8899. maintenance of low colonic pH (6). Butyrate, which is the preferred Email: [email protected] energy source for colonocytes (7), may beprotectiveagainst largebowel Colorectal cancer (CRC) is a major cause of death worldwide. cancer by inhibiting histone deacetylase thereby enhancing the apoptotic Studies suggest that dietary fibre offers protection perhaps by deletion of genetically damaged cells (8) through a histone hyperacety- increasing colonic fermentative production of butyrate. This lation-mediated pathway (9,10). Butyrate has also been shown to inhibit study examined the importance of butyrate by investigating the proliferation and induce differentiation of cancer cells in vitro (11,12). effects of resistant starch (RS) and butyrylated-RS on azoxyme- Strategies to increase colonic levels of SCFA, particularly butyrate, thane (AOM)-induced CRC in rats. Four groups (n 5 30 per may have clinical and public health benefits. However, there may be group) of Sprague–Dawley rats were fed AIN-93G-based diets limits to the usefulness of promoting RS consumption to increase containing a standard low-RS maize starch (LAMS), LAMS 1 butyrate levels as the microflora of some individuals have limited 3% tributyrin (LAMST), 10% high-amylose maize starch capacity to ferment certain types of RS, the SCFA profile produced (HAMS) and 10% butyrylated HAMS (HAMSB) for 4 weeks. can vary with RS source (13) and dietary fibre is not universally Rats were injected once weekly for 2 weeks with 15 mg/kg tolerated. Starches acylated to a degree of substitution of 0.2–0.25 AOM, maintained on diets for 25 weeks and then killed. Butyrate (i.e. 0.2–0.25 of the hydroxyl groups on each starch D-glucopyranosyl concentrations in large bowel digesta were higher in rats fed unit were derivatized or replaced by substituent acids) have been HAMSB than other groups (P < 0.001); levels were similar in shown to be an effective means of delivering specific SCFA to the HAMS, LAMS and LAMST groups. The proportion of rats de- colon in both animals (14,15) and humans (16). The SCFA, esterified veloping tumours were lower in HAMS and HAMSB than LAMS to the starch carrier molecule, are liberated by bacterial enzymes and (P < 0.05), and the number of tumours per rat were lower in are absorbed and utilized by the colonocytes or gut microbes. HAMSB than LAMS (P < 0.05). Caecal digesta butyrate pools This study investigated the importance of butyrate by examining and concentrations were negatively correlated with tumour size the effects of RS and butyrylated-RS on tumour development in rats (P < 0.05). Hepatic portal plasma butyrate concentrations were treated with azoxymethane (AOM). The effect of systemic butyrate higher (P < 0.001) in the HAMSB compared with other groups absorbed from the small intestine was also examined by the inclusion and negatively correlated with tumour number per rat (P < 0.009) of rats fed tributyrin (LAMST). and total tumour size for each rat (P 5 0.05). HAMSB results in higher luminal butyrate than RS alone or tributyrin. This is asso- Materials and methods ciated with reduced tumour incidence, number and size in this rat model of CRC supporting the important protective role of Animals and diets butyrate. Interventional strategies designed to maximize luminal Adult male Sprague–Dawley rats (198 ± 2 g) were purchased from the Animal butyrate may be of protective benefit in humans. Resource Centre, Murdoch University, Western Australia. The rats were housed in wire-bottomed cages in a temperature-controlled room (22–24C) with a 12 h light–dark cycle (lights on 8:00–20:00). They were allocated randomly into four groups (n 5 30 per group) with approximately equal body weights and given free access to water and diet throughout the study. Introduction The experimental diets were based on a control AIN-93G (17) diet that Colorectal cancer (CRC) is a common malignancy in Western socie- contained 530 g standard low-amylose maize starch, 200 g casein, 70 g corn oil and 50 g a-cellulose/kg of diet. The choline, methionine, minerals, vitamins ties resulting in 8% of all cancer deaths (1) and is emerging as a sig- and antioxidant were added as described previously (15). The amount of stan- nificant cause of morbidity and mortality in several Asian and Eastern dard maize starch in the diet was reduced to allow for the addition of exper- European countries as they adopt western dietary habits and lifestyles. imental starches and tributyrin. The experimental diets were control (LAMS, This cancer is considered mostly preventable by appropriate diet and low RS), low-amylose maize starch with 3% tributyrin (LAMST, low RS), 10% associated lifestyle factors (2). high-amylose maize starch (HAMS, high RS) or 10% butyrylated HAMS Resistant starch (RS) is dietary starch that escapes digestion in the (HAMSB, high RS). The latter was manufactured by National Starch Food small intestine. It is fermented by colonic bacteria with the production Innovation and had a degree of substitution of 0.25. HAMS and HAMSB were cooked with water and spray dried (to minimize crystallite formation) as de- scribed previously (18) before addition to the diets. This process was used to Abbreviations: AOM, azoxymethane; CRC, colorectal cancer; CSIRO, Com- mimic the condition of starches consumed in foods by humans as cooking monwealth Scientific and Industrial Research Organisation; HAMS, high- changes the production and delivery of butyrate to the colon by starches amylose maize starch; HAMSB, butyrylated high-amylose maize starch; LAMS, (15). All diets were prepared regularly at CSIRO and stored at 4C. The rats low-amylose maize starch; RS, resistant starch; SCFA, short chain fatty acids. were fed fresh diet daily. The Author 2008. Published by Oxford University Press. All rights reserved. For Permissions, please email: [email protected] The online version of this article has been published under an open access model. Users are entitled to use, reproduce, disseminate, or display the open access version of this article for non-commercial purposes provided that: the original authorship is properly and fully attributed; the Journal and Oxford University Press are attributed as the original place of publication with the correct citation details given; if an article is subsequently reproduced or disseminated not in its entirety but only in part or as a derivative work this must be clearly indicated. For commercial re-use, please contact [email protected] Resistant starch and butyrate on colon cancer in the rat After 28 days of feeding the experimental diets rats were injected with Caecal tissue and digesta weights were higher in rats fed HAMSB 15 mg of AOM/kg (Sigma Chemical Co., St Louis, MO). The rats were main- than all other groups (P , 0.001), and caecal tissue weight was high- tained on their experimental diets and were injected with the same dose of er in HAMS rats compared with LAMST (P , 0.01) (Table I). Caecal AOM a week later. They were monitored closely until the termination of the digesta pH was different between groups with pH in HAMSB-fed rats experiment 25–27 weeks post-initial AOM injection. Rats were anaesthetized lower than the other groups (P , 0.001), and the pH in the HAMS with halothane and blood collected into heparin-treated tubes from the hepatic group lower than the LAMS (P , 0.001) and LAMST (P , 0.05) portal vein. The small and large intestines and caecum were opened longitu- groups (Table I). The digesta pH fell from caecum to distal colon in all dinally, digesta collected and the epithelial surface cleaned with 0.15 mM groups except HAMSB; in the distal colon, the digesta pH of rats fed NaCl/l. The colon was divided into two equal length parts designated proximal LAMS was higher than HAMS (P , 0.01). and distal colon. The location, number and size of visible tumours were re- corded for each animal before the tumours were collected and fixed in 10% Caecal and proximal and distal colonic digesta butyrate concentra- buffered formalin, embedded in paraffin blocks and processed by routine his- tions were higher in rats fed HAMSB than other groups (P , 0.001) tological procedures for subsequent haematoxylin and eosin staining. and were similar in HAMS, LAMS and LAMST groups (Figure 1). All procedures involving animals were approved by the CSIRO Human Digesta SCFA pool sizes, calculated by multiplying the digesta wet Nutrition Animal Ethics Committee and complied with the Australian Code weights by the concentrations of each of the fatty acids measured of Practice (19). (acetate, propionate and butyrate), are shown in Table II. Digesta butyrate pool sizes were consistently larger throughout the large Analytical procedures bowel in rats fed HAMSB (P , 0.05–0.001). Caecal pools of acetate Analysis of SCFA, dry matter and pH was undertaken as described previously and propionate and proximate and distal colonic pools of propionate (15,20). All tumours were examined histologically by a single independent observer unaware of the dietary treatment and categorized into adenomas and were also larger in rats fed HAMSB (P , 0.001–0.05). Caecal and adenocarcinomas (invasive tumours) based on the criteria described previously distal pools of propionate were higher in HAMS than LAMST or (21). The ‘tumour incidence’ was defined as the proportion of rats in a group LAMS groups (P , 0.05–0.001), and proximal pools of propionate that had at least one tumour in the small or large bowel. The ‘size’ of each were higher in HAMS than LAMST (P , 0.001). tumour was calculated using the formula: Hepatic portal plasma butyrate concentration was higher (P , 0.001) in the HAMSB compared with the other groups (Figure 1). Dietary Tumour area index5p½ðD1  D2Þ=4 tributyrin (LAMST) increased hepatic portal butyrate concentration compared with LAMS to levels similar to HAMS (P , 0.001). where D and D are the length and width of each tumour. 1 2 More large bowel tumours were found in the distal colon rather Statistical analyses than the proximal colon (P , 0.02); however, a greater proportion of GraphPad Prism version 4.0 for Windows (http://www.graphpad.com) com- proximal tumours was adenocarcinomas (13 of 32) compared with the puter software was used for statistical analyses. Parametric data (body weight, distal colon, where most tumours were adenomas (55 of 67) organ weights and digesta measures) were analysed using one-way analysis of (P , 0.02). The size of caeco-colonic adenomas was positively and variance and Tukey’s post hoc multiple comparison test. Tumour indices were significantly correlated to the distance of the adenomas from the rectum compared between treatment groups using either Kruskal–Wallis test with (Spearman r 5 0.310, P , 0.008, n 5 74) with larger adenomas tend- Dunn’s post hoc tests or Mann–Whitney two-tailed t-tests for non-parametric ing to be closer to the caecum. The reverse was the case for adenocar- data. Tumour incidence data were analysed using Pearson’s v test using cinomas, with larger adenocarcinomas tending to be closer to the contingency tables. The relationships between butyrate and tumour measures rectum (Spearman r 5 0.403, P , 0.05, n 5 25). There was no were determined by non-parametric Spearman-ranked correlations. Data effect of diet on the distribution of tumours based on size or tumour are expressed as mean ± SEM with statistical significance indicated when P , 0.05. type i.e. the ratio of adenomas to adenocarcinomas in the proximal and distal colons was not significantly different between treatment groups. Large bowel tumour incidence (proportion of rats per group with Results tumours) was lower in HAMSB and HAMS compared with LAMS group (P , 0.05). Tumour number (number of tumours per group) Growth rates were similar in all the experimental groups and there was lower in HAMSB compared with LAMS (P , 0.05). There was were no significant differences in final body weights between the dietary treatments (mean for all groups 627 ± 7 g, Table I). Two rats were euthanased early due to events unrelated to the experimental procedures (from group LAMS in week 9; HAMSB, week 23). Seven rats were euthanased within 4 weeks of their scheduled dates because they showed possible symptoms of CRC including weight loss and rectal bleeding; tumour end points were obtained from these animals. Table I. Final measures of rats fed diets containing low-amylose maize starch (LAMS), LAMS with 3% tributyrin (LAMST), HAMS and butyrylated HAMS (HAMSB) LAMS LAMST HAMS HAMSB Body weight (g) 635 ± 15 621 ± 18 638 ± 14 616 ± 25 a a,b a Caecal tissue 0.84 ± 0.02 0.78 ± 0.02 0.93 ± 0.03 1.19 ± 0.03 weight (g) a a a Caecal digesta (g) 1.46 ± 0.07 1.41 ± 0.09 1.70 ± 0.09 2.27 ± 0.08 a,c a a ,d Caecal digesta pH 7.12 ± 0.04 7.07 ± 0.04 6.92 ± 0.03 6.69 ± 0.04 Distal colonic 6.97 ± 0.05 6.85 ± 0.07 6.66 ± 0.05 6.77 ± 0.06 Fig. 1. Concentration of butyrate in the digesta of the caecum, proximal and digesta pH distal colon (mM; n 5 28–30) and hepatic portal plasma (mM; n 5 26–30) of rats fed diets containing low-amylose maize starch (LAMS; open bars), Values are mean ± SEM, n 5 28–30. LAMS with 3% tributyrin (LAMST; light grey bars), (HAMS; dark grey Significantly different from HAMSB, P , 0.001. bars) and butyrylated HAMS (HAMSB; black bars) (mean ± SEM). b a b Significantly different from HAMS, P , 0.01. Significantly different from HAMSB, P , 0.001; significantly different c c Significantly different from HAMS, P , 0.001. from HAMS, P , 0.05 and significantly different from LAMST, P , 0.001 Significantly different from HAMS, P , 0.005 within each row of data. within each gut region or hepatic portal plasma. 2191 J.M.Clarke et al. an apparent trend towards reduced large bowel number for the HAMS particularly butyrate, to the colon (22). Low levels of colonic SCFA group compared with the LAMS (P , 0.055). Caecal butyrate pools in individual humans may result either from low consumption of di- and concentrations were significantly and negatively correlated with etary fibre or from an inadequate fermentative capacity of the colonic the number of large bowel tumours per rat (P , 0.03, r 5 0.206 and microflora. Earlier animal studies have shown that acylated starches P , 0.04, r 50.198, respectively), whereas there was no relation- are twice as effective as HAMS in raising large bowel SCFA levels of ship between distal butyrate levels and tumour number. The total the acid that has been esterified (14,15). The present data confirm tumour area for each rat was lower in the HAMBS than LAMS group those previous experiments and show that while both HAMS and (P , 0.05). The caecal butyrate pools and total large bowel tumour HAMSB raise total SCFA, the greatest increase in butyrate was with area for each rat were significantly and negatively correlated the butyrylated SCFA. HAMSB was particularly effective in raising (P , 0.05, r 5 0.185). Hepatic portal plasma butyrate concentra- butyrate concentrations in the distal colon, which is the site of most tion was negatively correlated with tumour number per rat (Spearman CRC in humans. r 5 0.243, P , 0.009) and total tumour area index for each rat This study examined the importance of butyrate by investigating (Spearman r 5 0.184, P 5 0.05). the effects of RS (a dietary fibre) and butyrylated-RS on levels of Most small intestinal adenomas were found in the proximal small butyrate in the large bowel and their impact on intestinal tumorigen- intestine (data not shown). There were no significant differences in the esis in an established animal model of CRC. The results from this incidence, numbers and ratio of adenomas to adenocarcinomas in the study are consistent with previous experiments showing that dietary small intestine between groups (Table III). Tumours in the small in- RS reduces CRC incidence in rats treated with AOM (23–26). The testine of rats fed LAMST were significantly larger than those in the importance of butyrate to this protection is demonstrated by the ability rats fed LAMS (P , 0.05) but the total small intestinal tumour area of an RS-equivalent HAMSB to achieve greater protection and higher per rat was not significantly affected by diet. butyrate levels. Statistical examination of the data showed that this effect correlated with caecal butyrate pools and concentrations and hepatic portal butyrate concentrations. Discussion The total tumour size per rat was also significantly lower in the Acylated starches were designed to promote large bowel health by HAMSB than LAMS group, which reflects a lower incidence and providing a vehicle for the rapid and sustained delivery of SCFA, smaller size of tumours in rats fed HAMSB. This further supports Table II. Digesta SCFA pools (micromoles) of rats fed diets containing low-amylose maize starch (LAMS), LAMS with 3% tributyrin (LAMST), HAMS and butyrylated HAMS (HAMSB) Diet Caecal SCFA pools Proximal colon SCFA pools Distal colon SCFA pools Acetate Propionate Butyrate Acetate Propionate Butyrate Acetate Propionate Butyrate a a,b a a a a,c a LAMS 107.7 ± 7.2 30.0 ± 1.8 21.3 ± 1.6 44.1 ± 4.2 11.4 ± 1.1 7.1 ± 0.7 40.9 ± 3.8 9.5 ± 0.8 7.8 ± 0.8 a a,b a b a,c a c a,c a LAMST 99.8 ± 7.6 27.8 ± 2.3 22.0 ± 1.7 31.6 ± 3.1 8.1 ± 0.7 5.3 ± 0.6 36.7 ± 4.1 8.7 ± 0.9 7.6 ± 1.0 a a a a a d d HAMS 132.2 ± 11.8 53.5 ± 4.6 36.2 ± 4.0 47.2 ± 4.8 17.2 ± 1.6 9.2 ± 0.9 55.4 ± 5.7 20.8 ± 2.1 12.8 ± 1.9 HAMSB 237.2 ± 26.8 125.7 ± 11.1 125.5 ± 12.5 45.5 ± 4.3 23.5 ± 2.3 18.7 ± 2.0 51.1 ± 5.0 29.5 ± 2.9 19.4 ± 2.0 Values are mean ± SEM, n 5 28-30 for caecal; 24–30 for proximal colon; 24–27 for distal colon. Significantly different from HAMSB, P , 0.001. Significantly different from HAMS, P , 0.05. Significantly different from HAMS, P , 0.001. Significantly different from HAMSB, P , 0.05 within each column of data. Table III. Indices of AOM-induced small and large bowel tumours in rats fed diets containing low-amylose maize starch (LAMS), LAMS with 3% tributyrin (LAMST), HAMS and butyrylated HAMS (HAMSB) (mean ± SEM) LAMS LAMST HAMS HAMSB Number of rats 29 30 30 29 Incidence of tumours (%) Small intestine 27.6 16.7 30.0 20.7 b c,d Large bowel 65.5 56.7 40 37.9 Number tumours per rat Small intestine 0.4 0.2 0.4 0.2 e c Large bowel 1.3 0.9 0.6 0.5 Average tumour area (tumour area index) Small bowel 35.9 ± 11.5 146.9 ± 70.5 49.0 ± 10.2 51.8 ± 11.0 Large bowel 26.2 ± 8.8 21.9 ± 4.6 49.5 ± 14.7 18.0 ± 5.1 Total tumour area per rat Small bowel 13.6 ± 5.8 29.4 ± 17.3 19.6 ± 6.9 10.7 ± 4.5 e c Large bowel 34.3 ± 12.7 19.8 ± 4.7 31.4 ± 12.7 9.3 ± 3.5 Tumour type (large intestine) Adenoma (number) 27 24 12 11 Adenocarcinoma (number) 11 3 7 4 a 2 Analysed using Pearson’s v test. Contingency comparison between two groups. Significantly different from HAMSB, P , 0.05. Significantly different from HAMS, P , 0.05. Analysed using Mann–Whitney non-parametric t-tests between two groups. Significantly different from LAMST, P , 0.05. 2192 Resistant starch and butyrate on colon cancer in the rat the argument that butyrate absorbed from the lumen of the large bowel cose are preferred ahead of SCFA as energy substrates by jejunal may have a role regulating the initiation and growth of large bowel enterocytes (38) that would normally be exposed to low-digesta tumours. butyrate levels, suggesting nutritional factors controlling cellular The relationship between tumour size and butyrate pool was stron- apoptosis may differ in the small and large intestine. Other workers ger for the caecum than the colon, which may reflect the large bowel have reported an increase in small intestinal tumour incidence, but physiology of the rat in which bacterial activity occurs principally in not size, in response to RS (39) in mice with a targeted mutant gene the caecum. resulting in spontaneous small intestinal tumours. In the AOM- There was no effect of tributyrin on tumour development in the treated rats in this study, HAMS did not increase small intestinal large bowel. Tributyrin (glycerol tributyrate) is absorbed in the small tumour size or incidence, supporting the epidemiological evidence intestine, raising free butyrate concentrations in peripheral plasma for that high-RS diets do not enhance the risk of tumourigenesis in the up to 4 h (27). Tributyrin has been recommended for clinical evalu- small intestine (40). ation as a possible treatment for leukaemia (28) but has not been The current study demonstrates that large bowel and hepatic portal found to reduce the incidence of AOM-induced CRC in mice (29). venous butyrate levels correlated negatively with tumour indices in an At the level of tributyrin included in the LAMST diet in this study accepted animal model of CRC. Butyrate was raised by HAMS rela- (3%), hepatic portal plasma butyrate concentrations were similar to tive to the LAMS control, but the highest butyrate levels were those that resulted from ingesting the HAMS diet. In a previous study achieved with HAMSB, indicating the greater potential effectiveness (unpublished data), hepatic portal butyrate concentrations up to 1.0 for this modified starch to significantly improve bowel health and mM were recorded in individual rats fed 3% tributyrin; however, these reduce the risk of developing CRC. Further work is necessary to de- levels were not associated with increased butyrate concentrations in termine the effectiveness of HAMSB in opposing initiation or pro- peripheral plasma. These data suggest that 3% tributyrin may not gression of experimental carcinogenesis. increase levels of systemically delivered butyrate to the large bowel and that butyrate absorbed from the small intestine in the rat is in- Acknowledgements effective in altering large bowel CRC induced by AOM. The authors wish to thank National Starch Food Innovation for providing Genomic instability is a prerequisite for oncogenesis and the gen- the HAMS and HAMSB and gratefully acknowledge Julie Dallimore, otoxic effects of AOM are thought to be responsible for tumour Ben Scherer, Jessica Winkler and Peter Royle for their contributions to the initiation leading to the development of aberrant crypts, adenomas study. In addition, the statistical advice of Dr Ian Saunders of CSIRO Math- and eventually invasive adenocarcinoma. Butyrate could inhibit this ematical and Information Sciences (Urrbrae, South Australia) was greatly process either by promoting apoptosis (8) or repair in damaged appreciated. colonocytes during the initiation phase or by suppressing the growth of cells with damaged DNA (30). Alternatively, butyrate may be Conflict of Interest Statement: None declared. reducing tumour growth by inhibiting hypoxia-induced angiogenesis (31,32). It is not clear which mechanism(s) is operating. Tumour numbers and incidence were lower in HAMSB than in LAMS. References The size of neoplastic lesions is an important factor clinically as 1. Garcia,M. et al. Global Cancer Facts & Figures 2007. American Cancer large adenomas are more likely to progress to adenocarcinomas Society. Atlanta, GA. (33). Progression to adenocarcinoma may be the consequence of 2. World Cancer Research Fund. 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Carcinogenesis – Pubmed Central

Published: Aug 13, 2008

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