Effect of resistant starch on genotoxin-induced apoptosis, colonic epithelium, and lumenal contents in rats

Richard K. Le Leu2, Ian L. Brown1, Ying Hu and Graeme P. Young{dagger}

Department of Medicine, Flinders University of South Australia, Bedford Park, 5042, Australia
1 National Starch and Chemical Company, Bridgewater, New Jersey, USA

2 To whom correspondence should be addressed Email: richard.leleu{at}flinders.edu.au


    Abstract
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
The effect of different doses of a type-2 resistant starch (RS) in the form of high amylose cornstarch (HAS) on the intralumenal environment and the acute-apoptotic response to a genotoxic carcinogen (AARGC) in the colon was assessed to determine if changes in lumenal conditions were associated with an enhanced apoptotic response to DNA damage. The control diet was a modified form of the AIN-76 diet containing fully digestible starch but no dietary fibre. HAS was added to the control diet at the expense of digestible starch to give 10% HAS, 20% HAS and 30% HAS. Rats were fed the different experimental diets for a period of 4 weeks, after which a single injection of azoxymethane was given to induce DNA damage in the colonic epithelium; 6 h later AARGC was measured. Other measures included fecal and cecal short chain fatty acids (SCFA) and pH, and cell proliferation in the colonic epithelium. In HAS-supplemented rats, fermentation events were significantly increased in both cecum and feces. There was a progressive decrease in pH in both the cecum and feces as the amount of HAS in the diet increased. SCFA concentrations, including butyrate, were significantly elevated by HAS with higher levels being observed in the cecum than in the feces. There was a significant increase in colonic AARGC with HAS doses of 20 and 30% (P < 0.01) but not with 10% HAS. Cell proliferation was not affected by any dose of HAS. Correlations with AARGC, independent of dietary group, were seen for fecal SCFAs and pH, suggesting that fermentation events, might explain the effect of RS on AARGC. Altering amounts of dietary RS changes fermentative activity in the colon. Increased RS is associated with enhanced AARGC. Changes in amount of fermentable substrate are capable of changing the biological response to DNA damage.

Abbreviations: ACF, aberrant crypt foci; AOM, azoxymethane; HAS, high amylose starch; RS, resistant starch; SCFA, short chain fatty acid


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Resistant starch (RS) is defined as the sum of starch and products of starch degradation that are not absorbed in the small intestine of healthy individuals (1). Therefore, RS reaches the colon undigested, similar to dietary fibre. RS in the colon is a potential source of fermentable substrate resulting in the production of gases (CO2, CH4, H2) and short chain fatty acids (SCFA) mainly acetate, propionate and butyrate (2,3). Intestinal fermentation of RS may have important implications for health, including protection against diarrhea (4) and colorectal carcinogenesis. Increased SCFA production, in particular butyrate, may offer protection (5). Butyrate, which is produced by anaerobic fermentation of carbohydrate and other substrates in the colonic lumen has been shown to inhibit cell proliferation, induce differentiation and enhance apoptosis in colorectal cancer cells in vitro (69). An epidemiological study showed a negative relationship between starch intake and colorectal cancer risk (10). It was hypothesized that the active component was RS and the mechanism for achieving colorectal cancer protection was through fermentation in the colon. Fermentation may be important in prevention of colorectal cancer via other effects as well (11). SCFA production lowers luminal pH, and bacterial 7-{alpha}-dehydroxylase activity, thereby reducing the conversion of primary to secondary bile acids, which are suggested to be colon tumor promoters (12).

RS can be classified into four main types, of which the first three may occur in a typical human diet (1). RS1 includes physically entrapped starch within whole plant cells and food matrices (e.g. coarsely milled grain). RS2 consists of native starch granules that are highly resistant to digestion by {alpha}-amylases (e.g. green banana, high amylose maize starch). RS3 comprises retrograded starches, formed when starchy foods are cooked and cooled. RS4 comprises chemically modified starches (e.g. esterified starches) where the modification interferes with the amylolytic activity of digestive enzymes.

The evidence from animal experiments of RS feeding on colorectal carcinogenesis is limited and conflicting (1318). An explanation for the varying results between the reported studies could be the different starch type and feeding regimens used, thereby altering lumenal contents. Conditions in the colonic lumen have a major influence on colonic oncogenesis (19).

The purpose of the present study was to investigate the effects of feeding increasing concentrations of RS2 [high amylose cornstarch (HAS)] to rats and assess effects on colonic lumenal environment along the length of the colon. In addition, we explored the relationship between changes in lumenal environment, namely SCFA concentration, fecal pH and fermentation, and certain epithelial events putatively related to colorectal cancer risk: specifically epithelial proliferation and the acute-apoptotic response to genotoxic carcinogens.


    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Animals and diets
A total of 96 male Sprague–Dawley rats, 5 weeks of age, were obtained from the Animal Resource Centre, Perth, Western Australia. Animals were divided randomly into four experimental groups and housed three per plastic cage in an animal holding room under controlled conditions of 22 ± 2°C (SD), 80 ± 10% humidity and 12 h light/dark cycle. Animals were given free access to water and weighed weekly throughout the study.

The diets were based on the AIN-76 standard for purified diets for rats and mice (20). Each group of animals was fed one of four diets (Table I). The first group ‘control’ consumed a diet containing no added fibre or RS. The second group ‘10% HAS’ contained 100 g/kg diet raw HAS. The third ‘20% HAS’ contained 200 g/kg diet. The fourth group ‘30% HAS’ contained 300 g/kg diet. HAS when raw has been shown previously to contain 61.8% RS (21) and in the present study was added to the diet at the expense of an equal amount of cornstarch. The HAS was supplied by National Starch and Chemical Company, Bridgewater, New Jersey, USA.


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Table I. Composition of experimental diets (g/100 g diet)

 
Experimental procedure
Fresh fecal samples were collected from each rat on the last 3 days of the experimental period by gently handling the rats until they produced a fecal sample. For fecal pH and SCFA analysis, the fresh feces were placed immediately in 2 ml of cold saline, homogenized, pH measured (TPB, digital pH meter, model 1852 mV, Brisbane, Australia) and stored at -20°C.

After 4 weeks on experimental diets each rat received a single i.p. injection of azoxymethane (AOM) (10 mg/kg body wt, Sigma Chemical Co., St Louis, MO) to induce apoptosis (22). The rats were also injected i.p. with vincristine sulfate (1 mg/kg body wt) 3 h before killing to arrest cells in metaphase of the cell cycle (23). Rats were killed by CO2-induced narcosis, 6 h after AOM treatment. Immediately after death, the entire colon was rapidly removed and divided into proximal and distal portions; the limit of the proximal portion was defined by the ‘herring bone’ pattern. These were flushed clean with ice-cold saline. Segments of 2 cm were taken from the cecal end of the proximal portion and the rectal end of the distal portion. These segments were placed in 10% buffered formalin for 24 h, then washed and stored in 70% ethanol. The cecum was excised, weighed and a known weight of content placed in 2 ml of cold saline for pH measurement and samples stored at -20°C for SCFA analysis.

The Flinders University of South Australia Animals Welfare Committee approved all experimental procedures.

Evaluation of apoptosis
Colon sections (0.5 x 0.5 cm) in 70% ethanol were cut from proximal and distal segments of the colon and embedded in paraffin. Paraffin-embedded sections (5 µm) were stained with hematoxylin and evaluated under a light microscope for apoptotic cells. Apoptotic cells were identified in 20 randomly chosen intact crypts by characteristic morphologic changes of: cell shrinkage, presence of condensed chromatin and sharply delineated cell borders surrounded with a clear halo (22) as described by Potten et al. (24). Apoptotic cells were clearly distinguished from cells undergoing metaphase arrest by the above characteristics (Figure 1). In all cases, an independent observer who was unaware of the experimental dietary treatment determined the quantification of apoptotic cells. The percentage of apoptotic nuclei (apoptotic index) was calculated as the mean number of apoptotic cells per crypt column divided by the total number of cells in the column and multiplied by 100. The length of each crypt was determined along with the position of apoptotic cells.



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Fig. 1. Photomicrograph (x40) of colonic crypts in rats showing apoptotic cells (A) and cells arrested in metaphase (M) 6 h after AOM injection and 3 h after vincristine sulfate injection that would be scored.

 
Determination of cell proliferation
To assess proliferation, paraffin-embedded sections (5 µm) were stained with hematoxylin. Twenty well-orientated and longitudinally sectioned crypts were examined for each rat. The total number of epithelial cell nuclei and the position and number of cells arrested in metaphase were recorded for each crypt and averaged for each animal. In all cases, an independent observer who was unaware of the experimental dietary treatment determined the quantification of cells arrested in metaphase. The mitotic index, which is calculated as the number of cells arrested in metaphase divided by the total number of cells in each crypt column multiplied by 100.

SCFA analysis
Fecal and cecal samples were homogenized in 4 vol of internal standard solution (heptanoic acid, 3.5 mM) and centrifuged at 3000 g for 10 min. The supernatant was then distilled and 0.3 ml injected into a gas chromatograph (Hewlett Packard 5890 Series II A) equipped with a flame ionization detector and a capillary column (Zebron ZB-FFAP, 30 m x 0.53 mm ID, 1 m film, SGE, Australia). Helium was used as the carrier gas, the initial oven temperature was 120°C and was increased at 20°C per min to 180°C, the injector temperature was 200°C and the detector temperature was 200°C. A standard SCFA mixture containing acetate, propionate and butyrate was used for calculation and the results are expressed as µmol/g of sample.

Statistical analysis
The distribution of each variable was assessed graphically to determine whether it was normally distributed. Those variables that were not normally distributed were subjected to logarithmic transformation before a significance test was performed. Differences between means were analysed by ANOVA and significance level was set at 0.05. Fisher's least significant difference test was used for multiple comparisons. The relationship between each of the cecal and fecal parameters and the apoptotic index of the epithelium in the distal colon was determined by the Pearson coefficient of regression model controlling for the effect of the different diets. Results are expressed as mean ± SE of the untransformed data. Statistical analyses were performed using SPSS version 10 software.


    Results
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Body weight
No significant difference was observed in body weight gain (g/week) between the rats fed the control-no fibre diet and the RS-supplemented diets (Table II).


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Table II. Effects of RS on weight gain, fecal pH, cecal weight, cecal content and cecal pH in rats1

 
Cecal fermentation
Cecal tissue weight significantly increased as the rats were fed increasing amounts of HAS (Table II). The wet weight of cecal contents followed the same pattern as cecal tissue weight, i.e. lowest in rats fed the control diet and highest in the rats fed the highest amount of HAS (Table II). Cecal pH was related inversely to the cecal tissue weight (r = -0.77, P < 0.001) and contents (r = -0.75, P < 0.001) and was lowest in rats fed the highest amount of HAS.

The results of SCFA analysis in the cecal content are expressed as µmol/g wet weight and are shown in Table III. The presence of HAS in the diet significantly increased total SCFA concentration in the cecum. The total cecal pool increased significantly in a dose-dependent manner as HAS supplementation increased in the diet. There was a 5-fold increase in rats fed the 30% HAS compared with those rats fed the control diet (P < 0.001). Supplementation with 20% HAS and 30% HAS significantly increased cecal acetate concentration (P < 0.05), with 30% HAS resulting in significantly higher acetate than all other treatments. Propionate concentration was enhanced with 20% HAS (P < 0.01); however, supplementation with 30% HAS resulted in a significant decline in cecal propionate concentration (P < 0.05). Butyrate was significantly elevated with all HAS doses (P < 0.05).


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Table III. Effects of RS on fecal and cecal SCFA concentrations in rats1

 
Fecal fermentation
Fecal pH was used as an indicator of distal lumenal pH (25) and is shown in Table II. A similar pattern was observed in the feces as to that in the cecal content. The pH levels declined as the HAS supplementation in the diet increased. The results of SCFA analysis in the feces are shown in Table III. Total SCFA concentration was significantly elevated in all HAS doses (P < 0.01). The 20% HAS dose resulted in significantly higher concentrations than all other HAS doses (P < 0.001). A similar pattern to that of total SCFA concentration was observed with fecal acetate concentration. Fecal propionate concentration was significantly elevated in the 10% HAS and 20% HAS doses (P < 0.01). Butyrate concentration in the feces was significantly elevated (P < 0.01) in all HAS doses.

Colonic epithelial responses
The acute-apoptotic response in the distal colon was significantly higher in the 20% HAS and 30% HAS doses compared with the control group (P < 0.01), with these increases in apoptotic index being 31.6 and 33.9%, respectively (Figure 2). The mean apoptotic index values ± SE were as follows: control 10.6 ± 0.4, 10% HAS 12.1 ± 0.9, 20% HAS 14.0 ± 1.0, 30% HAS 14.2 ± 1.2.



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Fig. 2. Effects of increasing amount of HAS on change in apoptotic index in the distal colon of rats treated with AOM. Apoptotic index is expressed as percentage relative to the control treatment group.

 
The proliferation rate of epithelial cells, as assessed by the number of cells arrested in metaphase per crypt column was not significantly different among the groups within the distal colon, nor was the mean crypt column height (Table IV).


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Table IV. Effects of RS on crypt column height and cell proliferation in ratsa

 
Association of cecal and fecal parameters with apoptosis in the distal colon
The apoptotic index of the distal colon was found to be significantly associated with a number of parameters in the cecum and feces after controlling for the effect of the different diets (Table V). The strongest relationships were seen between fecal SCFA (total and butyrate), pH and distal apoptotic index. In the cecum, strong relationships were seen between pH, total cecal pool and apoptotic index, while weaker relationships were seen with cecal acetate and total SCFA.


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Table V. Correlations between apoptotic index in the distal colon and selected fecal and cecal parameters

 

    Discussion
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Our study has shown that a diet enriched in HAS can significantly change the lumenal environment in a dose-dependent manner. Bacterial fermentation in the cecum and feces was markedly increased leading to higher levels of SCFA. Considerable acidification was observed also at these sites as well as a significant increase in the cecal tissue weight and cecal content.

Incorporation of as little as 10% HAS into the diet significantly changed parameters of fermentation. Higher concentrations of SCFA were observed in the cecum compared with the feces indicating that HAS is rapidly fermented. This is consistent with other studies examining RS (22,26) as rats are cecal fermentors and SCFA are rapidly absorbed in the large bowel. As the dose of HAS increased in the diet, greater fermentation activity was observed in the cecum. This was evidenced by increased cecal content and hypertrophy, greater SCFA concentrations and SCFA pool and lowered pH.

Despite observing an increased fermentation capacity in the cecum as the HAS dose increased to 30% of the diet, there was a decline in fermentation in the feces when HAS was increased from 20 to 30% of the diet. Morita et al. (27) reported that when a highly purified and digestible protein such as casein is the sole protein source in the diet in the presence of large amounts of fermentable carbohydrate such as HAS, nitrogen supply may become insufficient to sustain a rapid bacterial proliferation, thereby resulting in a decline in fermentation capacity. This may well explain the decline in SCFA production when the highest dose (300 g/kg) of HAS was incorporated in the diet. The lowest fecal pH was found in the rats fed the highest HAS dose despite SCFA concentration being lower than the 20% HAS. This may be explained by an accumulation of succinate (28), which has originated from an imbalance in the carbohydrate/nitrogen ratio through changes in the intestinal microflora.

Increased butyrate production is associated with induction of differentiation, suppression of proliferation and enhanced apoptosis in vitro (69) all of which might be beneficial. Higher butyrate levels may relate to lower tumour yield in rodent models (5,29). The present study showed that incorporation of as little as 10% HAS significantly increased butyrate concentration in the cecum and feces. The highest concentration in the feces was seen with 20% HAS although this was not significantly higher than the 10 and 30% HAS doses. Anti-carcinogenic effects associated with butyrate may be more applicable to the distal colon as that is where most tumors are seen. Wheat bran, which in contrast to other fibre sources may generate greater concentrations of butyrate in the distal colon and in most cases, is associated with protection against colorectal cancer (5,30,31). HAS in previous studies has been shown to be fermented rather rapidly (21,32), therefore it seems desirable to try and delay the fermentation of HAS so as to produce higher distal/fecal butyrate levels. The addition of a non-digestible protein source (i.e. potato protein) combined with HAS may help to sustain fermentation by correcting the imbalance in the ratio of carbohydrate/nitrogen in the colonic lumen (27) thereby delivering higher levels of butyrate to the distal colon. This needs to be tested further.

Spontaneous apoptosis in the distal colon of animals not receiving AOM has been shown previously to be very low (i.e. 0.5%) and does not appear to be regulated by the diet (34). In the present study we analysed the effect of HAS on the acute-apoptotic response to a genotoxic carcinogen (AARGC) in the distal colon. We found that HAS at 20 or 30% in the diet significantly facilitated AARGC by as much as 30%. Apoptosis is an important regulatory process in the protection against the development of cancer. Apoptosis acts by removing cells with genomic instability (33). Upregulation or facilitation of apoptosis during the initiation step of colorectal cancer may result in increased elimination of the DNA damaged cells that might otherwise progress to malignancy. There was no significant effect of HAS on cell proliferation within the distal colon thus increased AARGC was not due to increased DNA damage in S-phase resulting from increased proliferation rate exposing DNA to mutagen. Previous studies (22,34) showed that AARGC can be facilitated by dietary wheat bran and this effect may be linked to higher distal colonic butyrate levels. Interestingly, the magnitude of the increase in AARGC observed in the present study by 20 or 30% HAS is comparable with that achieved with wheat bran at a level of 5% total dietary fibre in the diet (22). Elevated distal butyrate levels have been linked to a protective value of wheat bran against development of colorectal cancer (5,14), although this association is not always seen (35). HAS significantly elevated butyrate levels in both the cecum and feces of the rats in the present study. When regression analysis was performed regardless of the dietary group, total fecal SCFA and the individual fecal SCFA as well as pH in the feces were significantly correlated with AARGC. This suggests that fermentation related events may be responsible for the increase in apoptosis.

The evidence from animal experiments of RS feeding on colorectal carcinogenesis is limited and conflicting (1318). One study reported that RS2 supplied as HAS at a level of 3 or 10% in the diet did not inhibit DMH-induced colon carcinogenesis in rats (13). However that particular study used an extremely high dosage of DMH (20 mg/kg body wt weekly for 20 weeks), which may have masked any effects of the RS in the diet. In another study (14), after 16 weeks of feeding a diet containing potato starch (RS2) there was a significant enhancement in the density of aberrant crypt foci (ACF). There was also an increase in the number and size of tumors per rat after 30 weeks. Two other rat studies utilizing ACF as the end point for oncogenesis have examined RS with mixed results. Cassand et al. (15) observed a fall in the total number of ACF/cm2 of colon in rats fed RS (type not reported) for 12 weeks when compared to a control RS-free diet. Retrograded HAS (RS3) however, when fed to rats for 4 weeks prior to initiation of ACF with DMH, did not affect ACF numbers (16). In Min mice, RS in the form of retrograded HAS (RS3) was shown not to influence the incidence or number of colon tumours (17) while RS2 feeding produced significantly more small intestinal tumours (18). Several explanations could explain the varying results between the reported studies: the different carcinogen protocols (dose and duration), the different starch type and feeding regimens used, thereby altering lumenal conditions. The conditions in the colonic lumen have a major influence on colonic oncogenesis (19). Fermentation of carbohydrates by intestinal microflora may lead to protection against CRC through secondary events such as fecal bulking, decreased transit time, acidification and production of SCFA, particularly butyrate (36).

Another explanation of how HAS may facilitate AARGC could relate to its prebiotic properties. A prebiotic is defined as a non-digestible food ingredient that beneficially affects the host by selectively stimulating the growth and/or activity of one or a limited number of bacteria in the colon that can improve the host health (37). RS has been shown to modify the composition of the fecal flora and selectively stimulate the growth of beneficial bacterial populations (3841). Hughes and Rowland (42) demonstrated that prebiotics such as inulin and fructo-oligosaccharide stimulate apoptosis in the colon of rats. Further studies are warranted to examine if certain bacterial species that are stimulated by prebiotics can modify the AARGC.

In conclusion, HAS significantly changes the lumenal environment through increased fermentation of RS. At a dose of 20 or 30% HAS in the diet, a significant elevation of AARGC in the distal colon was observed. Fermentation events seem a likely explanation for the effect of RS on apoptosis perhaps specifically due to butyrate. As the acute-apoptotic response to genotoxic carcinogens acts to remove genetically damaged cells that might otherwise form mutated clones that progress to malignancy, HAS might protect against the progression of mutated clones. To fully understand the effects of RS on oncogenesis it will be important to monitor lumenal changes in future studies. Those produced in these experiments might be those more likely to protect against oncogenesis.


    Notes
 
{dagger} Declaration of interest: G.P.Young is currently conducting research sponsored by National Starch and Chemical Company Inc. Back


    References
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 

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Received April 1, 2003; revised May 28, 2003; accepted May 29, 2003.