Stimulation of apoptosis by two prebiotic chicory fructans in the rat colon

R. Hughes and I.R. Rowland1

Northern Ireland Centre for Diet and Health, School of Biomedical Sciences, University of Ulster, Coleraine BT52 1SA


    Abstract
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Prebiotics, in particular the chicory derived ß(2-1) fructans, have been shown to exert cancer protective effects in animal models. The present study was carried out to determine the effects of two chicory fructans—oligofructose (RaftiloseP95; average degree of polymerization DP = 4) and long chain inulin (RaftilineHP; average DP = 25), on apoptosis and bacterial metabolism associated with carcinogenesis. Eighteen rats were fed a stock diet for one week. Three groups of six animals were then fed one of three diets: basal, basal with oligofructose (5%w/w) or basal with long chain inulin (5%w/w), for a three week period. All animals were then dosed with 1, 2-dimethylhydrazine and killed 24 h later. The mean number of apoptotic cells per crypt was significantly higher in the colon of rats fed oligofructose (P = 0.049) and long chain inulin (P = 0.017) as compared to those fed the basal diet alone. This suggests that oligofructose as well as the long chain inulin exert protective effects at an early stage in the onset of cancer, as the supplements were effective soon after the carcinogen insult. Comparison of the apoptotic indices between the two oligosaccharide diets showed no significant difference even though the mean apoptotic index was higher in animals fed long chain inulin. For all animals, apoptosis was significantly higher in the distal colon as compared to the proximal colon (P = 0.0002) however no significant site specific effect of diet occurred. There were no significant dietary effects on bacterial enzyme activities or ammonia concentration despite a trend towards increased colonic ß-glucosidase and reduced ammonia concentration during the oligosaccharide diets. This is the first time that a significant effect of chicory fructans on apoptosis has been shown and the results contribute to the growing evidence that chicory fructans may have cancer preventing properties.

Abbreviations: ACF, aberrant crypt foci; AI, apoptotic index; AOM, azoxymethane; DMH, 1,2-dimethylhydrazine; DP, degree of polymerization; H&E, haematoxylin and eosin; LAB, lactic acid-producing bacteria; NDO, non-digestible oligosaccharide; OF, oligofructose; SCFA, short chain fatty acid; TOS, galactooligosaccharide.


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
The functional effect of probiotics and prebiotics is a growing area of research. Species of the lactic acid producing bacteria (LAB) Bifidobacterium and Lactobacillus are the most widely studied probiotic strains and have been shown to exert cancer protective effects in vitro and in vivo (1,2). These organisms, have low activities of enzymes involved in the formation of genotoxic agents (e.g. ß-glucuronidase, azoreductase, nitro and nitrate reductases) compared with other major anaerobes in the gut (3). This may be one beneficial effect of dietary probiotics, assuming the organisms are able to establish themselves in the human large intestine at the expense of carcinogen metabolizing bacteria.

In humans, intestinal exposure to probiotics can occur via intake of foods containing these bacterial strains. Alternatively, increased levels of LAB in the intestine may be achieved by consumption of dietary substrates that are known to stimulate probiotic growth, i.e. prebiotics (4). Many non-digestible oligosaccharides (NDO) e.g. inulin, galactooligosaccharide (TOS) and oligofructose (OF) escape digestion in upper regions of the gastrointestinal tract and are known to exert prebiotic effects. For example, dietary supplements of TOS and OF increased intestinal bifidobacteria concentrations and suppressed faecal activities of carcinogen-metabolizing enzymes in humans and rats (5,6). In addition, lactulose which is a synthetic NDO, suppressed DNA damage in the colon mucosa of rats treated with 1,2-dimethylhydrazine (DMH) (7). Recently it has been shown that inulin at dietary concentrations of 5–10% suppressed azoxymethane (AOM) induced preneoplastic aberrant crypt foci (ACF) in rat colon (810). It was suggested that inulin is effective during the early promotion phase of cancer development as inulin suppressed ACF formation when given one week after a carcinogen dose (10). Interestingly, two of these studies showed a concomitant suppression of caecal ß-glucuronidase activity (8,10). This deconjugating enzyme is involved in DMH and AOM metabolism causing the release of the reactive metabolite methylazoxymethanol (MAM) from its biliary conjugate in the colon. Therefore, a decrease in ß-glucuronidase may be protective.

Although there are no epidemiological studies showing an effect of NDO consumption on colorectal cancer incidence, NDOs are found in a wide range of plant foods including onions, garlic and soybeans for which there is evidence of cancer protective activity (11). As well as modulating gut flora composition, NDOs may exert cancer protective effects at the cellular level following short chain fatty acid (SCFA) formation during fermentative bacterial metabolism. SCFAs i.e. butyrate, acetate and propionate, regulate colonic epithelial cell turnover and butyrate induces apoptosis in colon adenoma and cancer cell lines (12,13). In the colonic crypt, apoptosis maintains the balance in cell number between newly generated and surviving cells and at the luminal surface where differentiated epithelial cells are exfoliated (14). Butyrate formation from carbohydrate fermentation may explain the association between diet and apoptosis as shown by animal studies. Prolonged feeding of a high fat, low fibre Western diet to mice depleted the number of colonic apoptotic cells and this was associated with the development of gross lesions indicative of tumorigenesis (15). In contrast, a low risk Western diet i.e. low fat and high fibre, significantly increased carcinogen-induced colonic apoptosis in rats and reduced DNA damage as indicated by the Comet assay (16,17). Butyrate is produced during bacterial fermentation of inulin and oligofructose in vitro (4, 18) or in vivo in rats (19,20) at levels comparable to that produced from many non starch polysaccharides (21).

The aim of the present study was to investigate and compare the effects of oligofructose (RaftiloseP95) and long chain inulin (RaftilineHP) on apoptosis in the large intestine and on bacterial metabolic processes associated with carcinogenesis, i.e. ß-glucuronidase, ß-glucosidase activities and ammonia concentration. RaftilineHP consists of a mixture of linear ß(2-1) fructans with a degree of polymerization of 10–60 (average 22–25) and is derived from inulin following the removal of low molecular weight carbohydrates (fructose, glucose and DP2-10). RaftiloseP95 is mainly (>95%) oligofructose, the remainder comprises fructose, glucose and sucrose. RaftiloseP95 is produced by partial hydrolysis of inulin and contains smaller molecules with a degree of polymerization of 2–8 (average DP is 4.5). This variation in composition is reflected in a difference in fermentation rate. The longer chains being more slowly fermented than the short chains (22). Both supplements may therefore be metabolized at various sites in the large intestine, so dietary effects at proximal and distal regions of the large intestine were investigated.


    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Chemicals
1,2-Dimethylhydrazine was purchased from (Aldrich, Dorset, UK). General laboratory chemicals and most dietary ingredients were purchased from Sigma (Dorset, UK). Corn oil (Mazola) was obtained from a local retailer. RaftilineHP and RaftiloseP95 were supplied by Orafti (Tienen, Belgium).

Three diets were prepared for the study; a basal diet; basal with oligofructose and basal with the HP inulin. The basal diet was prepared in accordance to the American Institute of Nutrition 93 diet (AIN-93) (23) as shown in Table IGo. The test substances were added to the basal diet at a level of 5% (w/w) for each supplement. The diets were prepared in bulk prior to the study and stored at -20°C in 75 g portions. The portions were thawed at room temperature immediately before use.


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Table I. Composition of the purified basal diet.
 
Experimental procedure
Eighteen male Sprague–Dawley rats were obtained at 3–4 weeks of age and randomly assigned to six cages (three rats per cage). The animals were maintained under controlled environmental conditions and fed a stock laboratory rodent diet for 1 week. Each cage was then assigned to one diet (basal, oligofructose or the HP inulin) for a three week period. Body weights and food intakes were recorded twice weekly. At the end of the experimental period, each animal was administered DMH (20 mg/kg) dissolved in normal saline, by stomach gavage. All animals were killed 24 h later by carbon dioxide euthanasia. The colon was immediately removed from each animal and cut mid-way to separate the proximal and distal ends. Proximal and distal contents were then removed, pooled, weighed and diluted with 0.9% saline to yield a 20% (w/w) suspension. Samples were immediately frozen and stored at –20°C until analysis. 1 cm long regions of the colon tissues were taken, slit longitudinally and fixed in 10% neutral buffered formalin. The tissue was dehydrated by washing in a series of alcohol dilutions, embedded in paraffin wax and 5 µm through sections were cut. Two adjacent sections per slide were obtained for apoptosis analysis.

Assessment of apoptosis
Apoptotic cells were identified using a commercially available kit (ApopTag S7101, Appligene-Oncor, France). The wax sections were rehydrated through descending alcohol concentrations and protein was digested during a 15 min incubation with proteinase K. Endogenous peroxidase was removed by treatment with 2% hydrogen peroxide. The 3' hydroxy ends of broken DNA strands were enzymatically labelled with digoxigenin nucleotides. The DNA fragments were then allowed to bind to an anti-digoxigenin antibody bound to peroxidase. This antibody conjugate enzyme generates a permanent intense localized stain, allowing sensitive detection of apoptotic cells (Figure 1AGo). A negative control was performed for each section whereby water was substituted for terminal deoxynucleotidyl transferase to check for non-specific incorporation of nucleotides and non-specific binding of the enzyme conjugate. The presence of apoptotic cells was verified by checking cell morphology in hematoxylin and eosin (H&E) stained adjacent sections. Overall, more cells stained positively for apoptosis using the ApopTag kit. Only those cells which were identified as apoptotic in both ApopTag and H&E stained sections were counted. Characteristic morphological features used to identify apoptotic death included condensation of chromatin, condensation of cytoplasm, cell shrinkage and cytoplasm and nucleus fragmentation (13,14) (Figure 1BGo). Positively stained cells were counted in 20 good longitudinal sections of crypts. The number of positive cells expressed per crypt counted was termed the apoptotic index (AI).




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Fig. 1. Apoptotic cells in ApopTag and H&E stained sections from a rat colon. (A) Apoptotic cells (arrow) detected by in situ end-labelling (ISEL) using the ApopTag kit (magnification x200). The ApopTag kit stains apoptotic cells brown. (B) Apoptotic cells (arrow) in a H&E stained section (magnification x200).

 
ß-glucuronidase and ß-glucosidase activity and ammonia concentration
Colon suspensions were defrosted and centrifuged at 500 g for 3 min to remove bacterial debris. The supernatant was decanted and diluted 1:1 with anaerobic potassium phosphate buffer (final concentration 0.1 M) and used for enzyme analysis. Assays for ß-glucuronidase and ß-glucosidase were carried out at 37°C under an anaerobic atmosphere of 86%:10%:4% nitrogen: carbon dioxide: hydrogen. Each suspension was incubated with either p-nitrophenyl-ß-D-glucuronide or p-nitrophenyl-ß-D-glucopyranoside for ß-glucuronidase and ß-glucosidase analysis respectively. Aliquots of the reaction mixture were taken at specified timepoints and the release of p-nitrophenol was measured at wavelength 402 nm. Enzyme activities were expressed as µmol of product formed per hour per gram of colon contents. Ammonia concentration in the colon suspensions was determined as described by Wise et al. (24). Treatment with phenol nitroprusside and alkaline hypochlorite resulted in the development of a blue colour which was measured at 540 nm. Ammonia concentration was determined from a standard reference curve made up of different concentrations of ammonium chloride and expressed as µmol per gram of colon contents.

Statistical analysis
The data were log transformed (to the base 10) to achieve a normal distribution. The effect of diet on proximal, distal and total apoptosis and bacterial metabolism was tested using one-way ANOVA. F values with a probability <0.05 were regarded as significant. Tests of Least Significant Differences (LSD) were applied to compare means when a significant F value was determined. Regional differences in apoptosis were compared using one-way ANOVA.


    Results
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Body weight and colon contents
Samples from five animals from the basal group were available for analysis as one animal died within the 24 h period following carcinogen dose. There was no difference between the dietary groups in the amount of food eaten with all animals eating approximately 13 g of food per day. Mean body weight increased by 88 g (37–122 g) during the study period and there was no significant difference in rate of weight change between each dietary group (P = 0.56) (Table IIGo). Colon contents collected from the animals on the oligosaccharide diets were over twice the weight of those collected from animals consuming the basal diet. However these results did not reach statistical significance (P = 0.25) due to the small number of samples. Colon material was not present from two animals in the oligofructose group and in one of the five remaining rats in the basal diet group.


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Table II. Body weight change and weight of colon contents of rats fed oligofructose and inulin diets
 
Apoptosis
There was a significant effect of diet on the number of apoptotic cells per crypt (P = 0.045). Colonic apoptotic index results were higher in animals fed oligofructose and HP inulin as compared with animals fed the basal diet (P = 0.049, P = 0.017 respectively). Although mean AI was highest in rats fed the long chain inulin diet, the difference between the oligosaccharide diets was not significant (P = 0.53) (Figure 2Go). Collation of results from all animals showed that overall, apoptosis was more prevalent in the distal colon as compared to the proximal colon (P = 0.0002; Figure 3Go). When AI results from the proximal and distal regions were analysed separately, diet did not have a significant effect (P = 0.47, P = 0.19 respectively) even though AI in the NDO treated animals was markedly higher as compared to control animals (Figure 3Go).



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Fig. 2. Mean AI results. Error bars represent SEM. Rats were fed the diets shown for 3 weeks before being given DMH and killed 24 h later. Sections of colon tissue were prepared. The mean number of apoptotic cells per crypt for each section was recorded and expressed as the AI. Mean results with standard error are illustrated. *P < 0.05 in comparison with the basal diet (ANOVA).

 


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Fig. 3. Apoptotic indices in the proximal and distal colon. AI results per site are shown. Error bars represent standard error of mean.

 
ß-glucuronidase, ß-glucosidase and ammonia
Table IIIGo shows the mean changes in colonic enzyme activities and ammonia concentration during each diet. There was little change in colonic ß-glucuronidase activity with diet (P = 0.43) and while ammonia concentration decreased by approximately 40% in rats fed the oligofructose and HP inulin diets, the effects were not significant (P = 0.11). Colonic ß-glycosidase activity increased non significantly during the oligofructose and HP inulin diets as compared to results from the basal diet (P = 0.12 and P = 0.25 respectively).


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Table III. Colonic enzyme activities and ammonia concentration
 

    Discussion
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
The aim of the present study was to investigate a possible mechanism for the observed anticancer effects of chicory derived ß(1-2) fructans in laboratory animals. The dietary supplements were administered with a basal diet which conformed to the nutritional composition of the standard AIN-93 diet (23). The basal diet was supplemented with 249 g/kg corn oil so all diets provided 44% energy as fat. This level of fat intake is typical of a high fat Western diet which is often positively associated with colorectal cancer risk (25). All animals were dosed with DMH prior to being killed. This was included to increase the sensitivity of the assay as one of the immediate cytotoxic effects of DMH in the rat colon is upregulation of apoptosis (14,26) and suppression of proliferation (26). Cells which escape this apoptotic deletion following DMH treatment, may have the potential to give rise to tumours after a long period of latency (27). So, factors which increase apoptosis above levels induced by the carcinogen may reduce the chances of tumour formation. Results from the present study showed that apoptosis was significantly higher in the colons of rats fed oligofructose and HP inulin as compared to those fed the unsupplemented basal diet (P = 0.049 and P = 0.017 respectively). Both test substances increased apoptosis 24 h after treatment with DMH suggesting that they may be protective in the early stages of carcinogenesis. Other studies have reported protective effects of inulin at postinitiation stages in the form of suppression of early preneoplastic lesions following treatment with a carcinogen (9,10).

Although oligofructose and HP inulin differ in their chemical structures there was no significant difference in the apoptotic effects of both oligosaccharides despite a trend towards higher apoptosis in the colons of rats fed HP inulin. A trend of more potent anticancer effects of the long chain inulin was also reported in previous work (8). No significant site specific effects were evident even though mean results showed that the apoptotic indices were higher in the distal colons from both dietary groups. When data from each animal on each diet were combined, it was found that the apoptotic index was significantly higher in the distal colon as compared to the proximal colon (P = 0.0002). This regional effect is not surprising due to pre-treatment with DMH, which is known to target the distal colon causing tumour formation following prolonged treatment (28). A single treatment has been shown to induce apoptosis in a dose and region dependant manner in rats killed 24 h after treatment (16).

The increase in apoptosis did not appear to be a consequence of changes in intestinal metabolism of DMH since neither oligofructose nor inulin increased ß-glucuronidase activity. Previous studies have reported significant changes in bacterial enzyme activities and ammonia concentrations in rats fed inulin diets (9,10). The lack of significant findings in the present study may be due to differences in the source of rats used. Despite this result, there was a trend towards increased colonic ß-glucosidase activity and decreased ammonia concentration in the rats fed oligofructose or inulin. The small numbers of samples available particularly for ammonia analyses (Table IIIGo) most likely contributed to the lack of significant effects. Ammonia is a known tumour promoter (29) so suppression of ammonia formation in the gut can be considered beneficial. No relationship between ammonia and AI results could be studied due to low sample size. The increase in colonic ß-glucosidase activity during the oligosaccharide diets may represent a prebiotic effect as lactic acid bacteria have high levels of ß-glucosidase activities as compared to other members of the gut flora (3).

It is not possible to deduce the mechanisms by which oligofructose and HP inulin induce apoptosis from the present study as no significant positive associations were determined between apoptotic indices and other parameters measured despite trends towards lower ammonia and increased ß- glucosidase activity. Nevertheless, results support previous findings which show a positive effect of non digestible carbohydrates i.e. high fibre diets, on colonic apoptosis in animals (15,16). The formation of the fatty acid butyrate during microbial carbohydrate fermentation in the large bowel may explain this association as sodium butyrate induces apoptosis at physiological concentrations (2–4 mM) in colon adenoma and cancer cells (12,13).

Colonic weights were higher by about two fold in rats fed the oligosaccharide diets as compared with the basal diet although the large variation of the data resulted in the effects being non-significant (P = 0.56) (Table IIGo). Stool bulking is considered to be protective against colon cancer probably due to reduced exposure of the colonic mucosa to carcinogens (25). Inulin and oligofructose exert stool bulking effects in humans (30,31).

Even though the present study did not confirm previous studies showing significant effects of chicory fructans on bacterial enzymes and ammonia, an important significant positive effect on colonic apoptotic index was reported. This is the first time such an association has been shown. Although it was not possible to determine mechanisms for the upregulation of apoptosis, the findings from the present study support previous work showing a positive effect of other non digestible carbohydrates on colonic apoptosis. Induction of apoptosis may therefore provide a further contribution to the cancer protective effects of non digestible carbohydrates.


    Notes
 
1 To whom correspondence should be addressed Email: I.Rowland{at}ulst.ac.uk Back


    Acknowledgments
 
We thank Joy Mc Carron for her excellent technical assistance. The support of Orafti (Tienen, Belgium) is gratefully acknowledged.


    References
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 

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Received March 27, 2000; revised September 18, 2000; accepted September 18, 2000.