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Department of Food Science and Nutrition, University of Minnesota, St. Paul, MN 55108–6099
2To whom correspondence should be addressed.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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KEY WORDS: • colon cancer • bifidobacteria • oligosaccharide • probiotics • rats • oligofructose
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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, Reddy et al. 1974
). How the microflora
influence colon cancer risk is unknown. However, colonic bacteria
express enzymes known to be involved in the metabolism of
procarcinogens and tumor promoters. Thus, it is possible that changes
in the colonic bacterial populations could influence cancer risk by
altering the activity of these enzymes.
Gross taxonomic differences in the fecal microflora of humans have not
been found consistently between populations at high risk for colon
cancer compared with low risk populations (Finegold et al. 1974
, Moore and Holdeman 1975
). However,
Reddy (1990)
showed that diet, in the form of wheat bran
feeding, can reduce the activities of ß-glucuronidase, nitroreductase
and azoreductase, bacterial enzymes implicated in carcinogen
metabolism. Goldin and Gorbach (1984)
reported that
feeding humans Lactobacillus acidophilus also lowered fecal
ß-glucuronidase, nitroreductase and azoreductase activities,
suggesting that consumption of certain bacteria had a beneficial effect
on the balance of colonic bacteria.
The term probiotics has been coined to describe those bacteria that,
when consumed in a viable form, have a beneficial effect in some way on
the host. A number of studies in animal models demonstrate that
consumption of probiotic bacteria can reduce colon cancer risk.
Goldin and Gorbach (1980)
reported that feeding
Lactobacillus acidophilus to carcinogen-treated rats
both reduced tumor incidence and increased the latency period.
Bifidobacteria, another lactic acid–producing bacteria, have also been
investigated for their effect on colon cancer. Reddy and Rivenson (1993)
found that in rats treated with the
heterocyclic amine, 2-amino-3-methylimidazol[4,5-f]quinoline, to
induce colon cancer, inclusion of Bifidobacterium longum in
the diet at 0.5% reduced tumor incidence from 23% to 0. Several
studies have used colonic precancerous lesions, termed aberrant crypts,
as an index of cancer risk. These are focal lesions found in the colons
of carcinogen-treated animals. Aberrant crypts, described by
McLellan and Bird (1988)
, are characterized by a larger
size and thicker lining of epithelial cells than normal crypts. The
distribution of the aberrant crypts mirrors that of tumors, i.e., their
occurrence is primarily in the distal colon (Sandforth et al. 1988
). Furthermore, several studies have shown a high
correlation between the number of aberrant crypts and the number of
tumors that subsequently develop (Alabaster 1995
,
Shiyapurkar et al. 1992
). Kulkarni and Reddy (1994)
found that rats given the colon carcinogen azoxymethane
and fed diets containing 1.5% Bifidobacterium longum had a
43% reduction in the total number of aberrant crypts per colon
relative to a control diet containing no bifidobacteria. In another
study using dimethylhydrazine-treated rats, consumption of 6
x 109 bifidobacteria per day resulted in a
63% reduction in the number of aberrant crypts per colon
(Abdelali et al. 1995
). These studies indicate that
consumption of bifidobacteria reduces colon cancer risk in
carcinogen-treated animals, indicating that bifidobacteria are
acting as a probiotic.
Bifidobacteria are a predominant bacterial species in the colon of
humans. However, increasing the relative proportion of bifidobacteria
in the colon would appear to be of benefit, as suggested by the studies
above. Although consuming bifidobacteria appears effective in this
regard, there are issues of cost and maintenance of viability. In
vitro, growth of bifidobacteria can be selectively stimulated by
several carbohydrates indigestible by humans. In particular, the
fructans inulin and oligofructose seem effective at stimulating
bifidobacteria growth selectively in vitro (Wang and Gibson 1993
). Studies in humans have confirmed the ability of chicory
inulin and oligofructose to increase the proportion of bifidobacteria
in the stools (Gibson et al. 1995
, Hidaka et al. 1991
). Indigestible compounds that are capable of selectively
stimulating the growth of beneficial bacteria such as bifidobacteria
are now referred to as prebiotics.
Few studies of the effect of putative prebiotics on colon cancer risk
have been reported. Reddy et al. (1997)
examined the
effect of diets containing 10% oligofructose or inulin on the
development of aberrant crypts in azoxymethane-treated rats. Both
fructans reduced the number of aberrant crypts per colon, with inulin
being somewhat more effective than oligofructose. Thus, the evidence is
that consumption of both probiotic bacteria, particularly
bifidobacteria, and prebiotic indigestible carbohydrates such as the
fructans inulin and oligofructose reduces colon cancer risk in animal
models. This raises the question whether a combination of a probiotic
and a prebiotic, sometimes referred to as a synbiotic, will have an
additive or even synergistic effect on reducing colon cancer risk.
Although Koo and Rao (1991)
reported that a combination
of bifidobacteria and (oligofructose) Neosugar reduced aberrant crypt
number in carcinogen-treated mice compared with mice given neither,
their results do not allow conclusions regarding synergy of the two
factors because neither was fed individually.
This study had two objectives. The first was to investigate the effect of bifidobacteria and oligofructose, both separately and in combination, in amounts that would be attainable in a human diet, on the development of aberrant crypts in carcinogen-treated rats. The second was to examine the effect of different types of oligosaccharides against a background of bifidobacteria administration, on aberrant crypt development and the colonic cell proliferation rate.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Male Wistar rats (Harlan Sprague Dawley, Indianapolis, IN) were housed individually in suspended wire-bottomed cages. The initial body weight of the animals varied depending on the experiment. The care and treatment protocol was approved by the University of Minnesota Committee on Animal Care. Rats were fed the basal diet, a modification of the AIN-76A diet, for 3–5 d before the start of each experiment. The composition of the basal diet was as follows (g/kg): casein, 200; cornstarch, 450; sucrose, 150; corn oil, 100; cellulose 50; DL-methionine, 3; AIN-76A mineral mix, 35; AIN-76A vitamin mix, 10; choline bitartrate, 2; butylated hydroxytoluene (BHT),3 0.02; and menadione sodium bisulfite complex, 0.0013. The BHT and menadione sodium bisulfite were dissolved in the oil. In diets containing oligosaccharides, the oligosaccharides were added to a final concentration of 2% by whole diet dilution. The oligosaccharides used were oligofructose (OF; GTC, Westminster, CO), soybean oligosaccharide (SBO; Ajinomoto U.S.A., Teaneck, NJ), or wheat bran oligosaccharide (WBO; Megazyme, Warriewood, NSW, Australia), an arabino-xylan. Rats had free access to food and water at all times.
Experimental design.
One series of experiments was conducted to examine the effect of
dietary OF, bifidobacteria administration or a combination of the two
on the development of aberrant crypts. A second series examined the
effect of different oligosaccharides on aberrant crypt formation and
mucosal cell proliferation. The first series of experiments and
associated methods are described in detail in Gallaher et al. (1996)
. Briefly, rats were treated with the carcinogen
1,2-dimethylhydrazine (DMH) to induce aberrant crypt formation. Each
rat was gavaged with two doses of DMH (15 mg/kg) 1 wk apart. One week
after the second dose, the experimental treatments were begun. The
control groups were gavaged with 1 mL of skim milk per day and fed the
basal diet. Rats given bifidobacteria were gavaged daily with
108 bifidobacteria (Chr. Hansen, Milwaukee, WI) in skim
milk, except for Experiment 1, in which the dose of bifidobacteria was
109 daily. The length of treatments differed slightly with
each experiment, varying from 3.5 to 5 wk.
Analyses.
Aberrant crypts and aberrant crypt foci were enumerated on a 2 x 5 cm2 section of the distal colon, ~2 cm from the anal
end, by a modification of the method of Bird (1987)
.
Colonic mucosal proliferation rate was determined as the labeling index
of colonic crypts. The labeling index was determined as the proportion
of actively dividing cells per total number of cells in each crypt.
Immunohistologic detection of proliferating cell nuclear antigen (PCNA)
was used as the marker of actively dividing cells. Tissue sections of
each colon were mounted in paraffin and processed for the
immunohistologic determination of PCNA. Thin sections (4
µm) were cut and placed on polylysine-coated
slides and dried. Sections were deparaffinized in xylene and rehydrated
in 100% ethanol/95% ethanol. Endogenous peroxidase was quenched by
incubation in 0.1% H2O2, followed by rinsing
with water and PBS. Immunodetection of PCNA was achieved witih the use
of a commercial kit (an amplified biotin-streptavidin system,
Oncogene Science, Uniondale, NY). Darkly stained nuclei were assumed to
be in S-phase and were counted as positive. Between 8 and 21 whole
crypts were counted for each animal, in most cases 12 or more.
Statistical analysis.
Data were analyzed by ANOVA using SigmaStat for Windows version 1.0 (Jandel Scientific, San Rafael, CA). Differences among groups were analyzed by use of the Student-Newman-Keuls method for normally distributed data and by Dunn's method for nonnormally distributed data.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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. Groups administered either bifidobacteria or fed OF were present
in three of the six experiments. In the groups given bifidobacteria
only, two were numerically greater than the control in aberrant crypt
count and only one was less than the control. In the groups only fed
OF, two were essentially equivalent to the control and one was slightly
greater than the control. A group given bifidobacteria and fed OF was
present in all six experiments, and the number of aberrant crypts was
numerically lower than in the control group in five of six experiments.
The pattern for aberrant crypt foci was similar (data not shown).
However, only in Experiment 1 was this difference significant.
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). Thus, overall, it appeared that the combination of
administration of bifidobacteria and dietary OF significantly reduced
the number of aberrant crypts relative to the control group.
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).
We suggested that this relationship could explain the variability in
response seen in the different experiments we reported. We have since
conducted another experiment comparable to the previous ones.
Figure 3
shows the relationship between body weight at the time of
injection of the carcinogen and the median number of aberrant crypts in
the bifidobacteria + OF group as a percentage of the control. Each
point represents one experiment (n = 15–20 animals per
group). Medians were used in this plot (as opposed to means used in the
previous plot) because the data were not normally distributed. Further,
Experiment 1 was not plotted because the administered dose of
bifidobacteria was greater in that experiment than in the others
(109 vs. 108/d). The relationship now
appears exponential, with a high correlation
(r2 = 0.983, P < 0.02). Thus, the previously reported relationship continues to hold; as
body weight increases, the response to a treatment, as bifidobacteria +
OF, diminishes.
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shows the results of feeding OF, SBO and WBO in rats also given
bifidobacteria (108/d). There were no significant
differences among the groups by ANOVA. However, by Student's
t test, there was a highly significant difference between
the control group and the group fed WBO (P < 0.02). In
the second experiment (Fig.
5), OF, SBO and WBO were again fed, but a group given only
bifidobacteria was also included. Again, there were no significant
differences in aberrant crypt number among the groups by ANOVA.
However, there was a trend for a reduction in the bifidobacteria + OF group compared with the control group (P = 0.078 by
Mann-Whitney rank-sum test).
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shows the effect of bifidobacteria alone or of bifidobacteria
combined with feeding of OF, SBO or WBO, compared with the control
group. There were no significant differences among the groups byANOVA.
However, the bifidobacteria + SBO group had a significantly lower
labeling index than the control group by Student's t test
(P < 0.02). There was no significant correlation
between the median number of aberrant crypts and the mean labeling
index for each group (r = 0.20, P > 0.05).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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, Kulkarni and Reddy 1994
, Reddy and Rivenson 1993
, Reddy et al. 1997
). However, the ability of dietary oligofructose and
bifidobacteria to act in an additive or even synergistic manner has not
been established. Koo and Rao (1991)
found the
combination of bifidobacteria and (oligofructose) Neosugar to reduce
greatly the number of aberrant crypts in carcinogen-treated mice.
However, because neither bifidobacteria nor oligofructose was given
alone in that experiment, it was not possible to determine whether an
additive effect of the two agents occurred. In the series of
experiments reported here, bifidobacteria and oligofructose were given
alone and in combination in an attempt to determine whether an additive
effect occurred. In these experiments, dietary levels of oligofructose
and amounts of bifidobacteria that we believe might be attainable in
the human diet on an on-going basis were used, i.e., 2% dietary
oligofructose and 108/d of bifidobacteria. Other
investigators have used far greater amounts of oligofructose or
bifidobacteria, e.g. 10% oligofructose or 1.5% bifidobacteria in the
diet.
Our studies failed to show a significant reduction in aberrant crypt
number in the distal colon of rats fed either oligofructose or given
bifidobacteria alone (Fig. 1)
. However, in five of six experiments, the
combination of oligofructose and bifidobacteria led to a numerical
reduction in aberrant crypts compared with the control group given
neither. Although the reduction was significant in only one of these
experiments, a paired comparison of all six studies found a highly
significant difference between the control and bifidobacteria + OF
groups (Fig. 3)
. We believe that this indicates an additive, or
synbiotic effect of oligofructose and bifidobacteria for reduction of
colon cancer risk in carcinogen-treated rats. This is consistent
with a recent study by Rowland et al. (1998)
, who
reported that in azoxymethane-treated rats, consumption of either
inulin (Raftiline HPs 5%) or bifidobacteria (4 x
108/g diet) significantly reduced aberrant crypt
foci number (41 and 26%, respectively), and that the combination of
both reduced foci number by 80%, thus confirming a synergistic
synbiotic effect.
Although most recent studies of prebiotics have focused on the fructans oligofructose and inulin, other oligosaccharides may potentially be beneficial. In two separate experiments, we examined the effect of soybean oligosaccharide and wheat bran oligosaccharide, when given with bifidobacteria. To our knowledge, neither of these oligosaccharides has been examined previously as part of a synbiotic mix. In one experiment, both oligosaccharides produced a numerical reduction in aberrant crypts compared with the control group. However, this difference was significant only for the wheat bran oligosaccharide group. In a second experiment, there was little difference in aberrant crypt number between the control group and the groups fed these two oligosaccharides. Given our inconsistent results, the ability of other oligosaccharides to act as part of a synbiotic is uncertain.
Increased cell division, induced by nongenotoxic agents, has been shown
to be a cause of increased cancer risk (Preston-Martin et al. 1990
). The labeling index was used as an indicator of the cell
division rate within the colonic crypts. Immunohistologic detection of
proliferating cell nuclear antigen (PCNA), an auxiliary protein of DNA
polymerase 
, was used as the marker of actively dividing colonic
mucosal cells. Administration of bifidobacteria alone or in combination
with oligofructose or wheat bran oligosaccharide did not significantly
alter the labeling index. Only bifidobacteria with soybean
oligosaccharide reduced the labeling index significantly (P
< 0.02). We are not aware of other studies of either prebiotic
oligosaccharides or probiotic bacteria that have examined the colonic
labeling index. Pool-Zobel et al. (1996)
measured the
proportion of colonic cells positive for PCNA isolated from rats after
4 d of administration of different lactic acid bacteria, including
bifidobacteria. However, they reported only on PCNA expression in rats
given lactic acid bacteria, either with or without dimethylhydrazine
administration. Because their study did not include a group not given
lactic acid bacteria, their results cannot be compared with those
reported here.
Our results, using moderate levels of oligofructose and bifidobacteria,
strongly suggest that these two agents combined to fit the concept of a
synbiotic. Similar studies by Rowland et al. (1998)
,
using inulin as the prebiotic, confirm this concept. The ability of
other oligosaccharides to act as part of a synbiotic mixture is
uncertain. Finally, the lack of a correlation between the number of
aberrant crypts and the colonic crypt labeling index would cast doubt
on the significance of the cell division rate as a mechanism of
protection by the combination of oligosaccharides and bifidobacteria.
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FOOTNOTES |
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3 Abbreviations used: BHT, butylated
hydroxytoluene; bifido, bifidobacteria; DMH, 1,2-dimethylhydrazine; OF,
oligofructose; PCNA, proliferating cell nuclear antigen; SBO, soybean
oligosaccharide; WHO, wheat bran oligosaccharide.
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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