Carcinogenesis vol.22 no.1 pp.43–47, 2001
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
1To
whom correspondence should be addressed
Email: [email protected]
Prebiotics, in particular the chicory derived β(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 β-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.
Introduction
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
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, nondigestible oligosaccharide; OF, oligofructose; SCFA, short chain fatty acid;
TOS, galactooligosaccharide.
© Oxford University Press
genotoxic agents (e.g. β-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 (8–10). 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 β-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 β-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
43
R.Hughes and I.R.Rowland
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.
Table I. Composition of the purified basal diet.
Ingredient
Content g/kg diet
% total energy
Maize starch
Casein
Corn oila
Mineral mix (low calcium)
Vitamin mix
450
245
249
43
12.3
36
20
44
aContaining
dl-α-tocopherol acetate to provide 0.05 g/kg diet.
The composition of the vitamin and mineral mixes were as for AIN-.
93 with the exception that the mineral mix was reduced to 0.1%.
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. β-glucuronidase, β-glucosidase activities and ammonia
concentration. RaftilineHP consists of a mixture of linear
β(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
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
I. 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.
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
44
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 1A). 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 1B). 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).
β-glucuronidase and β-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 β-glucuronidase and β-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-β-D-glucuronide or p-nitrophenyl-β-D-glucopyranoside for
β-glucuronidase and β-glucosidase analysis respectively. Aliquots of the
reaction mixture were taken at specified timepoints and the release of pnitrophenol 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 oneway ANOVA.
Results
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 II). 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.
Anticancer effects of chicory fructans
Table II. Body weight change and weight of colon contents of rats fed
oligofructose and inulin diets
Diet
Basal
Oligofructose
HP inulin
Body weight change (g/day)
Colon contents (g)
Mean ⫾ SEM
n
Mean ⫾ SEM
n
0.82 ⫾ 0.1
1.15 ⫾ 50.1
1.11 ⫾ 0.2
5
6
6
0.413 ⫾ 0.1
0.92 ⫾ 0.3
0.99 ⫾ 0.2
4
4
6
Rats were fed the diets shown for 3 weeks before being given DMH and
killed 24 h later. The mean increase in body weight over the experimental
period is shown.
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).
Fig. 3. Apoptotic indices in the proximal and distal colon. AI results per
site are shown. Error bars represent standard error of mean.
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 ⫻200). The ApopTag kit stains
apoptotic cells brown. (B) Apoptotic cells (arrow) in a H&E stained section
(magnification ⫻200).
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
2). 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 3). 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
45
R.Hughes and I.R.Rowland
Table III. Colonic enzyme activities and ammonia concentration
Diet
Basal
Oligofructose
HP inulin
β-glucuronidase µmol/h/g
β-glucosidase (µmol/h/g)
Ammonia (µmol/g)
Mean ⫾ SEM
n
Mean ⫾ SEM
n
Mean ⫾ SEM
n
79.23 ⫾ 18.9
60.54 ⫾ 39.4
70.40 ⫾ 13.2
4
3
6
2.3 ⫾ 70.8
7.10 ⫾ 1.9
4.99 ⫾ 2.5
3
3
6
80.61 ⫾ 3.5
48.88 ⫾ 15.62
47.03 ⫾ 7.8
3
2h
4
Rats were fed the three diets for 3 weeks and then given a dose (p.o.) of DMH (20 mg/kg) and killed 24 h later. Suspensions of colon contents were prepared
and enzyme activities and ammonia concentration measured.
respectively) even though AI in the NDO treated animals was
markedly higher as compared to control animals (Figure 3).
β-glucuronidase, β-glucosidase and ammonia
Table III shows the mean changes in colonic enzyme activities
and ammonia concentration during each diet. There was little
change in colonic β-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 β-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).
Discussion
The aim of the present study was to investigate a possible
mechanism for the observed anticancer effects of chicory
derived β(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
46
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 β-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 β-glucosidase activity and decreased ammonia concentration in the rats fed oligofructose or inulin. The small
numbers of samples available particularly for ammonia
analyses (Table III) 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 β-glucosidase activity during the oligosaccharide diets may represent a prebiotic effect as lactic
acid bacteria have high levels of β-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 βglucosidase 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 II). 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
Anticancer effects of chicory fructans
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.
Acknowledgements
We thank Joy Mc Carron for her excellent technical assistance. The support
of Orafti (Tienen, Belgium) is gratefully acknowledged.
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Received March 27, 2000; revised and accepted September 18, 2000
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