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Differential effects of dietary whey, casein and soya on colonic DNA damage and large bowel SCFA in rats fed diets low and high in resistant starch

Published online by Cambridge University Press:  01 March 2007

Shusuke Toden ,
Anthony R. Bird ,
David L. Topping  and
Michael A. Conlon
Shusuke Toden
Affiliation:
Food Futures National Research Flagship, CSIRO Human Nutrition, PO Box 10041, Adelaide BC SA 5000, Australia Discipline of Physiology, School of Molecular Science, University of Adelaide, Adelaide, SA 5005, Australia
Anthony R. Bird
Affiliation:
Food Futures National Research Flagship, CSIRO Human Nutrition, PO Box 10041, Adelaide BC SA 5000, Australia
David L. Topping
Affiliation:
Food Futures National Research Flagship, CSIRO Human Nutrition, PO Box 10041, Adelaide BC SA 5000, Australia
Michael A. Conlon*
Affiliation:
Food Futures National Research Flagship, CSIRO Human Nutrition, PO Box 10041, Adelaide BC SA 5000, Australia
*
*Corresponding author: Dr M. A. Conlon, fax +61 8 8303 8899, email [email protected]
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Abstract

Feeding higher levels of dietary animal protein (as casein or red meat) increases colonic DNA damage and thins the colonic mucus barrier in rats. Feeding resistant starch (RS) reverses these changes and increases large bowel SCFA. The present study examined whether high dietary dairy (casein or whey) or plant (soya) proteins had similar adverse effects and whether dietary RS was protective. Adult male rats were fed diets containing 15 or 25 % casein, whey or soya protein with or without 48 % high amylose starch (as a source of RS) for 4 weeks. DNA damage was measured in isolated colonocytes using the comet assay. Higher dietary casein and soya (but not whey) increased colonocyte DNA damage. DNA damage was highest with soya when fed at 15 or 25 % protein without RS. Dietary RS attenuated protein-induced colonocyte DNA damage in all groups but it remained significantly higher in rats fed 25 % soya compared with those fed 15 % protein. Dietary protein level did not affect colonic mucus thickness overall but the barrier was thinner in rats fed high dietary casein. This effect was reversed by feeding RS. Caecal total SCFA and butyrate pools were higher in rats fed RS compared with digestible starch. Caecal and faecal SCFA were unrelated to genetic damage but correlated with mucus thickness. The present data confirm that higher dietary protein affected colonocyte DNA and colonic mucus thickness adversely but that proteins differ in their effects on these indices of colon health. The data show also that these changes were reversed by RS.

Keywords

CaseinWhey proteinSoya proteinResistant starchColonic genotoxicity
Type
Full Papers
Information
British Journal of Nutrition , Volume 97 , Issue 3 , March 2007 , pp. 535 - 543
Copyright
Copyright © The Authors 2007

Diet is implicated strongly in the pathogenesis of serious large bowel diseases. Recent large population studies have found that high intakes of red and/or processed meat are positive risk factors of colo-rectal cancer (Cassidy et al. Reference Cassidy, Bingham and Cummings1994; Rieger et al. Reference Rieger, Parlesak, Pool-Zobel, Rechkemmer and Bode1999; Norat et al. Reference Norat, Lukanova, Ferrari and Riboli2002, Reference Norat, Bingham and Ferrari2005; Chao et al. Reference Chao, Thun, Connell, McCullough, Jacobs, Flanders, Rodriguez, Sinha and Calle2005; Larsson et al. Reference Larsson, Rafter, Holmberg, Bergkvist and Wolk2005). The components of these meat-based foods responsible for increased risk remain to be established. It has been suggested that minor meat components such as haem iron contribute to this increased risk. In a meta-analysis of prospective population studies, Cassidy et al. (Reference Cassidy, Bingham and Cummings1994) identified dietary protein per se as an independent risk factor for colo-rectal cancer. High protein intakes have other adverse effects on bowel health as evidenced by an increased likelihood of relapse for ulcerative colitis patients (Jowett et al. Reference Jowett, Seal, Pearce, Phillips, Gregory, Barton and Welfare2004). Inflammatory bowel disease is a risk factor for large bowel cancer (Chen et al. Reference Chen, Rabinovitch, Crispin, Emond, Bronner and Brentnall2005), supporting a role for the protein component in disease aetiology.

We have shown previously that high dietary levels of animal-derived proteins (casein and red meat) increased colonocyte DNA damage in rats (Toden et al. Reference Toden, Bird, Topping and Conlon2005, Reference Toden, Bird, Topping and Conlon2006). Genomic damage is a prerequisite for cancer and the data could help to explain the associations between the consumption of red meat and meat products and large bowel cancer (Norat et al. Reference Norat, Bingham and Ferrari2005). Both protein types also induced a thinning of the mucus barrier in the colon which could be a potential contributory factor in inflammatory bowel disease (Shaoul et al. Reference Shaoul, Okada, Cutz and Marcon2004). However, the fact that high dietary levels of casein had the same effects as red meat suggest that the effect was not specific to one type of protein. Our knowledge of the role of dietary proteins in cancer aetiology in general is poor and it remains to be established whether other protein types have similar effects. For example, whey protein consumption is generally regarded as being protective against experimental colo-rectal cancer (Belobrajdic et al. Reference Belobrajdic, McIntosh and Owens2003; Xiao et al. Reference Xiao, Badger and Simmen2005) and also can reduce aberrant crypt foci compared with other protein types (McIntosh & Le Leu, Reference McIntosh and Le Leu2001; Belobrajdic et al. 2003). However, it is not known whether its actions are affected by dietary inclusion level. The situation with soya is confused with several contradictory experimental studies on colon cancer risk and soya protein consumption (Govers et al. Reference Govers, Lapre, De Vries and Van der Meer1993; McIntosh et al. Reference McIntosh, Regester, Le Leu, Royle and Smithers1995; Hakkak et al. Reference Hakkak, Korourian, Ronis, Johnston and Badger2001b; Badger et al. Reference Badger, Ronis, Simmen and Simmen2005). Isoflavones and other phytochemicals are often present in soya-based products and have been shown to inhibit cancer cell growth and induce apoptosis (Hakkak et al. Reference Hakkak, Korourian, Ronis, Johnston and Badger2001b). However, several rodent studies showed that soyabean protein increased colo-rectal cancer risk, induced damage to the colonic epithelium and stimulated colonocyte proliferation when compared with a casein diet (Govers et al. Reference Govers, Lapre, De Vries and Van der Meer1993; McIntosh et al. Reference McIntosh, Regester, Le Leu, Royle and Smithers1995). Soya protein isolates have been investigated extensively also for their ability to lower plasma cholesterol relative to casein (Lin et al. Reference Lin, Meijer, Vermeer and Trautwein2004), again suggesting some differences in nutritional properties.

Intake of fruits, vegetables and grains is linked to reduction of colo-rectal cancer risk (Potter et al. Reference Potter, Slattery, Bostick and Gapstur1993; Giovannucci & Willett, Reference Giovannucci and Willett1994; Steinmetz & Potter, Reference Steinmetz and Potter1996). These foods contain non-starch polysaccharides (NSP; major components of dietary fibre) which are thought to contribute to their protective action. Many also contain resistant starch (RS) which can also contribute to their protective effect (Topping & Clifton, Reference Topping and Clifton2001). RS is that fraction of starch which enters the large bowel undigested (Englyst et al. Reference Englyst, Wiggins and Cummings1982) and increases colonic fermentation and large bowel and faecal SCFA concentrations (Cummings & Macfarlane, Reference Cummings and Macfarlane1991). Butyrate, a major SCFA, is the preferred energy source of colonocytes (Clausen & Mortensen, Reference Clausen and Mortensen1995; Ritzhaupt et al. Reference Ritzhaupt, Ellis, Hosie and Shirazi-Beechey1998) and appears to provide protection against activities associated with carcinogenesis (Lupton, Reference Lupton1995). Furthermore, epidemiological studies have related a reduced risk of colon cancer to increased consumption of starch (Cassidy et al. Reference Cassidy, Bingham and Cummings1994; Bingham et al. Reference Bingham, Day and Luben2003). Our previous studies (Toden et al. Reference Toden, Bird, Topping and Conlon2005, Reference Toden, Bird, Topping and Conlon2006) also showed that dietary RS protected against casein and red meat-induced colonic DNA damage and some loss of the mucosal barrier. The previous findings provide support for a role of RS in maintaining colonic integrity and lowering the risk of serious large bowel disease. However, not all experimental studies support the protective effects of RS. One study reported that lipid peroxidation-induced DNA damage was increased in colonic mucosa of health subjects when they were fed the high RS diets (Wacker et al. Reference Wacker, Wanek, Eder, Hylla, Gostner and Scheppach2002). Another study showed that RS increased intestinal tumour formation of Apc1638N mouse model compared with those fed the control diets (Williamson et al. Reference Williamson, Kartheuser, Coaker, Kooshkghazi, Fodde, Burn and Mathers1999).

The question of the differential effects of animal and plant proteins is an important one and remains open. In the present study, we have sought to examine the effects of three different types of dietary proteins, casein, whey and soya, on colonic DNA damage, colonic mucus barrier thickness and SCFA levels in the caecum and faeces of rats. In addition, we have examined the ability of dietary RS to modulate the effects of changes in the colon induced by these proteins.

Materials and methods

Animals and diets

Adult male Sprague-Dawley rats (n 96) weighing approximately 200 g were obtained from the Animal Resource Centre, Murdoch University, Perth, Western Australia. Rats were housed in wire-bottomed cages in a room with controlled heating and lighting (20–23°C with a 12 h light–dark cycle) and had free access to food and water. They were allocated randomly to twelve groups (eight per group) and fed one of twelve diets (Table 1) for 4 weeks. Each cage had a ceramic bowl to ensure that the animals had access to a surface other than wire mesh.

Table 1 Composition of experimental diets (g/kg diet)*

*Diets based on AIN-93 (G) formulation.

The diets, which were based on the AIN-93G diet formulation, contained 15 or 25 % casein (New Zealand Milk, Auckland, New Zealand), whey isolate (Balance Muscle Technologies, Auckland, New Zealand) and soya isolate (Phyto Foods Australia Pty Ltd, NSW, Australia), with or without 48 % high amylose maize starch (Hi-maize™; National Starch and Chemical Company, Sydney, NSW, Australia) as a source of RS. Diets without high amylose starch contained highly digestible starch (Cornstarch 3401C; National Starch and Chemical Company). All diets contained 5 % wheat bran as the fibre source.

All procedures involving animals were approved by the Commonwealth Scientific and Industrial Research Organisation (CSIRO) Health Sciences and Nutrition Animal Ethics Committee and the University of Adelaide Animal Ethics Committee and conformed to published guidelines (National Health and Medical Research Council, 2004).

Sampling procedures

In the final 4 d of the experiment rats were placed in metabolic cages to collect faecal output. At the conclusion of the study the rats were anaesthetized with 4 % halothane/oxygen and gut tissues and digesta were collected as described previously (Toden et al. Reference Toden, Bird, Topping and Conlon2005). Faecal and caecal digesta samples were frozen for subsequent analyses.

Comet assay

A 6 cm segment of colon was removed from each rat at a point 3 cm from the most distal end of the colon and colonocytes were isolated immediately. These cells were used for the measurement of DNA strand breaks using the single-cell gel electrophoresis (comet) assay as described previously (Anderson et al. Reference Anderson, Hambly, Yu, Thomasoni and Shuker1999). Comet tail moment is the product of tail length and the fraction of DNA in the tail and was calculated for fifty cells from each of three slides per rat. Tail moment was calculated by Scion Image Beta 4.02 image processing and analysis software (Scion Corp., Frederick, MD, USA) using a public domain macro (Helma & Uhl, Reference Helma and Uhl2000).

Colonic mucus layer thickness

A 1 cm segment of colon was removed from each rat at a point 2 cm from the most distal end of the colon and cut open along the anti-mesenteric ridge and the mucosal surface was washed gently with 0·15 m-NaCl solution to remove digesta. The thickness of mucus lining the colon was determined by further cutting the tissue into 1·6 mm lengths, illuminating the tissue segments, capturing numerous images of the mucus layer along each segment, and then measuring the thickness using an image analysis program as described previously (Toden et al. Reference Toden, Bird, Topping and Conlon2005). For each animal, ten measurements were taken at different points along four tissue segments to give forty thickness measurements in total.

SCFA

Faecal and caecal contents were thawed then distilled and homogenised with 20 μl 1·68 mm-heptanoic acid (internal standard). The contents were analysed for SCFA (acetic, propionic and butyric acids), in duplicate, using an Agilent Technologies 6890N Network Gas Chromatograph System fitted with a Zebron ZB-FFAP column (0·53 mm × 30 m) (Phenomenex, Torrance, CA, USA) as described previously (Patten et al. Reference Patten, Abeywardena, McMurchie and Jahangiri2002).

Statistical analyses

Data are presented as means and their standard errors for each treatment group except in tables (where they are presented with pooled standard error values). The effect of RS and protein, and their interactions, were determined by three-way ANOVA and differences between treatments were analysed post hoc by Tukey's test. The relationship between caecal and faecal variables, colonic DNA damage and mucus layer thickness was determined by regression analysis. Data underwent logarithmic transformation when needed. Analyses were performed using a Statistical Analysis Systems statistical software program (version 8.02: SAS Institute, Cary, NC, USA). A value of P < 0·05 was used as the criterion of significance.

Results

Body weight gain and daily food and water intakes

The mean initial live weight for all groups combined was 241 (sem 2) g. Neither live weight gain (data not shown) nor final body weight differed between the experimental diets (mean final weight 353 (sem 4) g) over the 4-week study. There was no effect of protein type, protein level or RS on the daily food intake of the animals (Table 2). Neither RS nor protein type affected water intake but there was an independent effect of protein type and an interactive effect of protein level and type on daily water intake (both P < 0·05). Water consumption was highest in rats fed the 25 % soya diet.

Table 2 Effects of dietary casein, whey, soya and high amylose starch maize (resistant starch, RS) levels on daily food and water intakes, faecal and urinary outputs, and caecal and faecal measurements in rats* (Mean values with their the pooled standard error, n 8)

a–e Mean values within a row with unlike superscript letters were significantly different (P < 0·05).

* For details of procedures, see p. 537. Data were analysed by three-way ANOVA and post hoc analysis by Tukey's test.

Caecal content and tissue weights, faecal and urine output, intestinal weight and length

The wet weight of caecal content in RS-fed rats was 5·1 times higher than those fed standard starch (P < 0·001; Table 2). There was also a significant interaction between protein type and RS. Caecal tissue weight was unaffected by protein type or level but was significantly greater in rats fed RS (Table 2). There was also an interaction between dietary protein type and RS with a significantly lower weight in rats fed 25 % whey and RS compared with the other groups fed RS. Caecal digesta pH values were greater than 7 in all rats fed the standard starch diets with the exception of those fed the 25 % soya diet (Table 2). There was a small effect of protein type (but not level) on pH and a significant interaction between protein type and level and between these effects and RS.

Faecal output was unaffected by dietary protein level but was significantly higher in all groups of rats fed RS (Table 2). The type of dietary protein interacted with RS with a significantly lower output of faeces in rats fed 25 % casein with RS compared with other groups fed the high RS diet. The pH of faeces followed the same trend as the caecal values (i.e. >7) in rats fed the low RS diet with significantly greater acidity in rats fed RS. There were effects of protein level and type with the highest pH (close to 8) in rats fed whey and standard starch.

Daily urine output was unaffected by RS (Table 2) but was affected significantly by protein level with higher outputs in all groups fed 25 % protein.

There were increases in both weight and length of the colon and small intestine when rats were fed the RS compared to those on diets without RS (all P < 0·001; Table 3). However, protein type did not influence weight or length of either the colon or small intestine independently, but did interact significantly with RS.

Table 3 Effects of dietary casein, whey, soya and high amylose starch maize (resistant starch, RS) levels on length and weight of the colon and small intestine and weight of colonic contents in rats* (Mean values with their pooled standard error, n 8)

a–d Mean values within a row with unlike superscript letters were significantly different (P < 0·05).

* For details of procedures, see p. 537. Data were analysed by three-way ANOVA and post hoc analysis by Tukey's test.

DNA damage

The viability of colonocytes isolated for the comet assay was 92·2 (sem 2·4) % (all groups combined) and did not differ significantly between groups. When RS was absent from the diet, comet tail moments of isolated colonocytes were approximately twice as great for 25 % casein diets compared with those for 15 % casein diets (P < 0·001; Fig. 1). Similarly, the 25 % soya diet increased the tail moment by 87 % compared to the 15 % soya diet (P < 0·05). However, there was no significant difference between 15 and 25 % whey diets which had tail moments similar to the 15 % casein and soya groups. Overall, rats fed whey protein showed less colonic DNA damage compared to those fed casein or soya (P < 0·001). Inclusion of RS in the diet lowered the level of colonic DNA damage for all three dietary groups (P < 0·001). Although RS reduced soya-induced colonic DNA damage, colonocyte comet tail moment of rats fed 25 % soya with RS remained significantly greater than those fed the 15 % soya diet.

Fig. 1 Effects of dietary casein (■, 15 %; ■, 25 %), whey (, 15 %; □, 25 %), soya (▨, 15 %; ▥, 25 %) and resistant starch (RS) levels on colonic genetic damage in rats. The comet assay was performed on colonocytes extracted from the colon and the resulting comet tail moments (comet tail length × % DNA in the tail) are presented. For details of procedures, see p. 537. Values are means with their standard errors depicted by vertical bars (n 8). a–f Mean values with unlike superscript letters were significantly different (P < 0·05). Significant main effects of the dietary groups are RS level (P < 0·001), protein type (P < 0·001), protein level (P < 0·001), protein type × protein level (P < 0·01) and protein level × RS (P < 0·001).

Colonic mucus layer thickness

The colonic mucus layer was thicker in rats fed RS compared to those fed standard maize starch (P < 0·001). The 25 % casein diet reduced colonic mucus layer thickness by 62 % compared to the 15 % casein diet in rats consuming low RS (P < 0·001), but there was no similar reduction of mucus thickness in rats fed the high soya and whey diets with low RS (Fig. 2). The inclusion of RS in the diet prevented the loss of mucus thickness induced by high casein. Regression analysis indicates that there is a significant inverse relationship between comet tail moment of colonocytes and mucus thickness (n 96, r − 0·41, P = 0·001).

Fig. 2 Effects of dietary casein (■, 15 %; ■, 25 %), whey (, 15 %; □, 25 %), soya (▨, 15 %; ▥, 25 %) and resistant starch levels on the thickness of the mucus lining the colon. For details of procedures, see p. 537. Values are means with their standard errors depicted by vertical bars (n 8). a–d Mean values with unlike superscript letters were significantly different (P < 0·05). Significant main effects of the dietary groups are RS level (P < 0·001), protein type × RS (P < 0·01) and protein level × RS (P < 0·05).

Caecal and faecal SCFA

Caecal and faecal acetate, butyrate, propionate and total SCFA pools were all increased by dietary RS (Table 4). Protein type also affected both caecal and faecal acetate, propionate, butyrate and total SCFA pools (Table 4) and rats fed RS showed significantly lower caecal and faecal pH (P < 0·001; Table 2). There was a significant correlation between caecal and faecal pH with respective total SCFA pools (caecal r − 0·81, P < 0·0001; faecal r − 0·67, P < 0·0001). DNA damage as measured by the comet assay was found to be significantly associated with several caecal and faecal parameters. The significant relationships seen with DNA damage were with acetate pools (caecal r − 0·33, P < 0·05; faecal r − 0·35, P < 0·05), butyrate pools (faecal r 0·34, P < 0·05) and total SCFA pools (caecal r − 0·402, P < 0·001; faecal r − 0·36, P < 0·05). In addition, a significant relationship was seen with mucus layer thickness and total SCFA pools (caecal r 0·39, P < 0·001; faecal r 0·40, P < 0·001).

Table 4 Effects of dietary casein, whey, soya and high amylose starch maize (resistant starch, RS) levels on faecal and caecal SCFA pools in rats* (Mean values with their pooled standard error, n 8)

a–g Mean values within a row with unlike superscript letters were significantly different (P < 0·05).

* For details of procedures, see p. 537. Data were analysed by three-way ANOVA and post hoc analysis by Tukey's test.

Discussion

The present data confirm and extend our previous investigations on the effects of dietary protein level and RS on the colonic environment in rats. Specifically, we have shown again that increasing levels of dietary casein from 15% to 25% induces greater DNA strand breakage and thinning of the mucus layer. Up until now, we have only studied proteins of animal origin but these experiments show that a plant protein concentrate (soya) caused even greater colonocyte DNA damage than casein. Collectively, the data suggest that the basic effect was due to the protein content of the diet, rather than minor components such as haem (in meat) or phytochemicals (in soya). However, this does not explain the lack of a genetic damaging effect of higher dietary whey protein. A recent study showed that dietary exposure to both whey and soya proteins altered colonic global gene expression profiles, including repression of a key regulatory gene for cell cycle progression in rats with azoxymethane-induced colon tumours (Xiao et al. Reference Xiao, Badger and Simmen2005). In addition, previous rodent studies, using colon tumour induction with azoxymethane or 1,2-dimethylhydrazine, showed that whey protein was protective compared to casein, red meat and soyabean diets (Hakkak et al. Reference Hakkak, Korourian, Ronis, Johnston and Badger2001a; McIntosh & Le Leu, Reference McIntosh and Le Leu2001; Belobrajdic et al. Reference Belobrajdic, McIntosh and Owens2003). Another study demonstrated that bovine lactoferrin, a major whey component, reduced colon carcinogenesis in the post-initiation stage in azoxymethane-treated rats, with significant reduction of adenocarcinoma incidence (Tsuda et al. Reference Tsuda, Sekine, Ushida, Kuhara, Takasuka, Iigo, Han and Moore2000). A suggested protective mechanism of whey is its capacity to increase tissue glutathione. Whey protein has been shown to raise hepatic and (to a lesser extent) colonic glutathione concentrations in rats (McIntosh et al. Reference McIntosh, Regester, Le Leu, Royle and Smithers1995) which may facilitate deactivation of xenobiotics via glutathione transferase activities (Bounous, Reference Bounous2000). Animal studies have shown that a fraction of dietary protein (resistant protein) escapes into the large bowel in a manner analogous to RS (Morita et al. Reference Morita, Kasaoka, Ohhashi, Ikai, Numasaki and Kiriyama1998). A loss of protein from the small intestine has been reported in human subjects with ileostomy (McBurney et al. Reference McBurney, Thompson, Cuff and Jenkins1988). However, there seems to be no difference in the ileal digestibilities of cheese and beef protein which were 86–89 % (Silvester et al. Reference Silvester, Bingham, Pollock, Cummings and O'Neill1997). This suggests that differences between proteins in the present study were due to other causes.

Another finding which has been replicated is attenuation of the DNA-damaging effect of casein by RS (Toden et al. Reference Toden, Bird, Topping and Conlon2005). Dietary fibre is important for large bowel function and studies in rodents with experimentally induced cancer have suggested that cereal fibres may be protective (Reddy, Reference Reddy2000). However, human studies have given quite contradictory findings. A large, multicentre prospective European study found a substantial (30 %) reduction in relative risk from the lowest to the highest strata of intake (Bingham et al. Reference Bingham, Day and Luben2003). In contrast a meta-analysis of a number of prospective studies failed to find any protective effect of fibre (Asano & McLeod, Reference Asano and McLeod2002). The expectations of a beneficial effect of fibre came from observations of low risk of colo-rectal cancer in populations who were consuming unrefined foods which were assumed to be high in dietary fibre. Subsequent analysis has shown some of these diets to be actually relatively low in fibre but high in RS (Topping & Clifton, Reference Topping and Clifton2001) leading to higher large bowel SCFA. It appears highly possible that protection may be conferred by RS rather than NSP. The present data support this proposal as does the recent observation of a protective effect of RS against experimental cancer in rats (Bauer-Marinovic et al. Reference Bauer-Marinovic, Florian, Muller-Schmehl, Glatt and Jacobasch2006).

Despite the reduction with inclusion of RS, higher levels of DNA damage were found in rats fed soya. The present data are consistent with the increased colonic epithelial cell damage and increased proliferation with rats fed soya protein compared to rats fed casein (Govers et al. Reference Govers, Lapre, De Vries and Van der Meer1993) and also the increased tumour incidence and burden of dimethylhydrazine-induced intestinal tumour in rats compared to those fed whey or casein (McIntosh et al. Reference McIntosh, Regester, Le Leu, Royle and Smithers1995). The present experimental data are contrary to meta-analyses that suggest that there is an inverse correlation between consumption of non-fermented soya with colo-rectal cancer (Wu et al. Reference Wu, Yang and Pike2000; Spector et al. Reference Spector, Anthony, Alexander and Arab2003). The reasons for this difference are unclear as are those for the generally greater levels of damage with soya seen in this experiment. Minor soya components (e.g. saponins) are known to increase bile acid and steroid excretion (Lee et al. 2005) and secondary bile acids are thought to be cytotoxic within the large bowel (Narisawa et al. 1974).

The mechanism for the reduction of strand breaks by RS may lie in greater large bowel SCFA availability, especially of butyrate. While SCFA in general are thought to promote a favourable colonic environment, butyrate is thought to be especially important for mucosal integrity both as a metabolic fuel for colonocytes and also through specific actions which promote a normal cell phenotype (Rieger et al. Reference Rieger, Parlesak, Pool-Zobel, Rechkemmer and Bode1999; Emenaker & Basson, Reference Emenaker and Basson2001). The sampling regime of the present experiment precluded the direct measurement of colonic SCFA. However, we did determine caecal and faecal SCFA as indirect measures of production and colonic exposure, respectively. As anticipated, the feeding of RS raised caecal SCFA, consistent with greater fermentation. No consistent relationship was found between the pools of total and individual SCFA and genetic damage. For example, SCFA and butyrate pools were low in rats fed casein and whey in the absence of RS. However, butyrate pools were significantly higher in those fed soya at both levels, i.e. where damage was greater. Similarly, both caecal total SCFA and butyrate were higher in rats fed RS independent of comet tail moment. The present data are similar to those noted earlier in man (Schwiertz et al. Reference Schwiertz, Lehmann, Jacobasch and Blaut2002; Muir et al. Reference Muir, Yeow, Keogh, Pizzey, Bird, Sharpe, O'Dea and Macrae2004) and animals (Le Leu et al. Reference Le Leu, Hu and Young2002; Annison et al. Reference Annison, Illman and Topping2003; Bird et al. Reference Bird, Flory, Davies, Usher and Topping2004) with various types of RS. Using faecal SCFA as an index of colonic exposure there was a similar lack of relationship between genetic damage and total and individual SCFA. However, faecal SCFA may not be an entirely appropriate measure of exposure as SCFA excretion can be affected by transit as well as production (Topping & Clifton, Reference Topping and Clifton2001). Inter alia the present data support an interaction between undigested protein, RS and large bowel SCFA (Morita et al. Reference Morita, Kasaoka, Ohhashi, Ikai, Numasaki and Kiriyama1998; Toden et al. Reference Toden, Bird, Topping and Conlon2005). The present data also suggest that the interactions between dietary protein, RS and genetic damage are complex. Other fermentation products and compounds present in the diet may have had a role in damage and/or repair (Rowland et al. Reference Rowland, Mallett and Wise1985; Smith & Macfarlane, Reference Smith and Macfarlane1996). In addition, previous studies have suggested that carcinogenic N-nitroso compounds, often found in large quantity in red and processed meat, may also have a strong role in damaging large bowel environment (Cross & Sinha, Reference Cross and Sinha2004). Finally, there is a further interaction which remains to be explored, i.e. the microflora. The RS fed in the present experiment is known to be a prebiotic (Topping et al. Reference Topping, Fukushima and Bird2003) and has also been shown to modify the activity of pathogenic organisms in vitro (Kleessen et al. Reference Kleessen, Stoof, Proll, Schmiedl, Noack and Blaut1997) and so could have led to changes in genetic damage and repair through modifying bacterial activity.

As noted previously (Toden et al. Reference Toden, Bird, Topping and Conlon2005), the mucus barrier was thinner in rats fed higher dietary casein in the absence of RS. A similar loss has been seen with high red meat diets (Toden et al. Reference Toden, Bird, Topping and Conlon2006) but was not observed with soya or whey. Feeding RS prevented this loss with casein and led to a generally thicker barrier. Previous studies suggest that SCFA increase colonic mucus secretion (Cummings & Macfarlane, Reference Cummings and Macfarlane1991; Shimotoyodome et al. Reference Shimotoyodome, Meguro, Hase, Tokimitsu and Sakata2000). Mucins, the main component of mucus, are encoded by MUC genes and several MUC genes are speculated to have links with colo-rectal cancer. Recently, Willemsen et al. (Reference Willemsen, Koetsier, van Deventer and van Tol2003) used cell culture supernatant to demonstrate that SCFA differentially regulates prostaglandin production to stimulate mucin secretion via MUC2 expression in intestinal epithelial cells. However, it is possible also that the microflora may have interacted with diet to affect mucus thickness. Large bowel bacteria are known to possess mucinase activity (Dwarakanath et al. Reference Dwarakanath, Campbell, Tsai, Sunderland, Hart and Rhodes1995) and alterations in this capability could have affected barrier thickness. This is also a potential explanation for the differential effects of whey and soya as neither of these led to the loss of mucus seen with casein. The reversal of the thinning of the mucus barrier by dietary protein by RS warrants further investigation. Loss of barrier function is a feature of inflammatory bowel disease and higher dietary protein has been related to loss of remission from ulcerative colitis (Jowett et al. Reference Jowett, Seal, Pearce, Phillips, Gregory, Barton and Welfare2004). RS has been shown to protect against experimental colitis (Moreau et al. Reference Moreau, Martin, Toquet, Laboisse, Nguyen, Siliart, Dumon and Champ2003). While doubts have been cast on the relevance of this finding, there is an opportunity to determine the role of interactions between RS and other dietary components in this type of large bowel dysfunction.

The high and low ileal digestibility of the standard and high amylose maize starches, respectively, has been confirmed in rats with an effective ileostomy (Morita et al. Reference Morita, Kasaoka, Hase and Kiriyama1999, Reference Morita, Tanabe, Takahashi and Sugiyama2004). The differences in large bowel variables with RS (greater digesta and faecal mass, lower pH, greater caecal tissue weight) reflect this difference and accord with previous findings (Topping et al. Reference Topping, Gooden, Brown, Biebrick, McGrath, Trimble, Choct and Illman1997; Toden et al. Reference Toden, Bird, Topping and Conlon2005). Lower digesta pH is consistent with SCFA production leading to direct acidification and also utilisation of nitrogen (as NH4+) for bacterial growth. Faecal pH values reflected those in the caecum, being lower in rats fed RS with values closer to 8 in rats fed the highly digestible starch. Lowering of pH is a possible contributor to some of the changes in genetic damage through reducing the bioavailability of potentially toxic biogenic agents. However, while this factor could have contributed to some of the observed effects, it was not a prime determinant as there was no significant relationship between pH and either genetic damage or mucus layer thickness.

As anticipated from previous studies (Topping et al. Reference Topping, Gooden, Brown, Biebrick, McGrath, Trimble, Choct and Illman1997; Toden et al. Reference Toden, Bird, Topping and Conlon2005), the mass of large bowel tissue was higher in rats fed RS than in those consuming the highly digestible starch. The greater length of the colon is also consistent with findings from pigs fed RS (Topping et al. Reference Topping, Gooden, Brown, Biebrick, McGrath, Trimble, Choct and Illman1997). These changes have been explained through a greater availability of SCFA as metabolic fuels.

In conclusion, we have demonstrated that casein, whey and soya proteins have differential effects on colonic DNA damage. Importantly, none of the effects were due to any differences in food intake or body weight. A moderately high level (25%) of whey in the diet did not significantly increase the colonic DNA damage in comparison to 15 % whey. Moreover, increased dietary casein and soya, particularly in the absence of RS and its fermentation products, appear to increase DNA damage in a rodent model. It appears likely that dietary proteins interact differently with RS in the colon because the SCFA differed among protein types when fed with RS. However, we have shown that including RS in the diet reduces colonic DNA damage regardless of the types of protein consumed. Further investigations are necessary to ascertain whether the proteins we have examined and RS exert similar effects in man.

Acknowledgements

The authors thank Dr Pat Buckley for helpful discussions.

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Figure 0

Table 1 Composition of experimental diets (g/kg diet)*

Figure 1

Table 2 Effects of dietary casein, whey, soya and high amylose starch maize (resistant starch, RS) levels on daily food and water intakes, faecal and urinary outputs, and caecal and faecal measurements in rats* (Mean values with their the pooled standard error, n 8)

Figure 2

Table 3 Effects of dietary casein, whey, soya and high amylose starch maize (resistant starch, RS) levels on length and weight of the colon and small intestine and weight of colonic contents in rats* (Mean values with their pooled standard error, n 8)

Figure 3

Fig. 1 Effects of dietary casein (■, 15 %; ■, 25 %), whey (, 15 %; □, 25 %), soya (▨, 15 %; ▥, 25 %) and resistant starch (RS) levels on colonic genetic damage in rats. The comet assay was performed on colonocytes extracted from the colon and the resulting comet tail moments (comet tail length × % DNA in the tail) are presented. For details of procedures, see p. 537. Values are means with their standard errors depicted by vertical bars (n 8). a–f Mean values with unlike superscript letters were significantly different (P < 0·05). Significant main effects of the dietary groups are RS level (P < 0·001), protein type (P < 0·001), protein level (P < 0·001), protein type × protein level (P < 0·01) and protein level × RS (P < 0·001).

Figure 4

Fig. 2 Effects of dietary casein (■, 15 %; ■, 25 %), whey (, 15 %; □, 25 %), soya (▨, 15 %; ▥, 25 %) and resistant starch levels on the thickness of the mucus lining the colon. For details of procedures, see p. 537. Values are means with their standard errors depicted by vertical bars (n 8). a–d Mean values with unlike superscript letters were significantly different (P < 0·05). Significant main effects of the dietary groups are RS level (P < 0·001), protein type × RS (P < 0·01) and protein level × RS (P < 0·05).

Figure 5

Table 4 Effects of dietary casein, whey, soya and high amylose starch maize (resistant starch, RS) levels on faecal and caecal SCFA pools in rats* (Mean values with their pooled standard error, n 8)