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Department of Applied Biochemistry, Hiroshima University, Higashi-Hiroshima 739-8528, Japan;
*
Iida Women’s Junior College, Nagano 395-0812, Japan;
Gifu City Women’s College, Gifu 501-0192, Japan; and
**
Research Institute for Radiation Biology and Medicine, Hiroshima University, Hiroshima 734-0037, Japan
1To whom correspondence should be addressed. E-mail: nkato{at}hiroshima-u.ac.jp.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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KEY WORDS: • Buckwheat protein • 1–2-dimethylhydrazine • colon carcinogenesis • cell proliferation • rats
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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,2)
, although its digestibility is
relatively low (3)
. Our experimental studies with rats
have shown that a buckwheat protein product
(BWP)2
has hypocholesterolemic (4
,5)
, anticonstipation
(6)
and antiobesity activities (7)
and
chemopreventive activity against mammary tumorigenesis
(8)
. In addition, consumption of BWP reduces
gastrointestinal transit time in rats (3)
. These functions
appear to be due to its lower digestibility. The physiologic functions
of dietary BWP appear to be similar to those of some dietary fibers
(9
,10)
.
Some dietary fibers suppress the development of colon cancer
(11)
, and an epidemiologic study showed an association
between the risk of colorectal cancer and protein intake
(12)
. Indigestible protein has been reported to have an
important role through its interaction with resistant starch in
providing short-chain fatty acids (SCFA) to colonic tissue
(13)
, leading to improvement of large bowel physiology
(14)
. We proposed that consumption of BWP might suppress
the development of colon cancer due to its lower digestibility. It has
been reported that dairy proteins protect against
dimethylhydrazine-induced intestinal cancers in rats
(15)
. However, data concerning the influence of dietary
protein on colon carcinogenesis remain limited. Thus, we investigated
the effect of dietary BWP on the development of 1,2-dimethylhydrazine
(DMH)-induced colon cancer in rats.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Preparation of the buckwheat protein product (BWP) was described
(5)
. The composition of BWP was as follows (g/kg): water,
18; protein, 722; lipids, 210; starch, 17 (measured by a kit;
UV-method for determination of starch, Boehringer Mannheim,
Mannheim, Germany); crude fiber, 2. The amino acid composition of
casein and BWP determined using an amino acid analyzer is described
elsewhere (4)
. The compositions of fatty acids and sterols
in the lipid of BWP were described in our previous study
(5)
.
Animals and diets.
Male Sprague-Dawley rats (5 wk old, CD:Crj) were purchased from
Charles River Japan (Hino, Japan) and housed individually in metal
cages in a room with controlled temperature (24 ± 1°C) and a
12-h light:dark cycle (lights on, 0800–2000h); they had free access
to the diets and deionized water. The rats were maintained according to
the guidelines established by Hiroshima University. After consuming
commercial stock diet for 1 wk, the rats (average 101 g) were
divided into two groups (n = 20/group). The
composition of experimental diets is presented in Table 1
. The net concentration of protein in the diet was 200 g/kg. The dietary
level of fat was adjusted to a final concentration of 100 g/kg by
adding corn oil (Table 1)
. The feeding period was 124 d. Food
consumption and body weight were recorded daily. Feces were collected
for the final 3 d of the experimental period. Rats were given DMH
(Nacalai Tesque, Kyoto, Japan, 10 g/L of 0.1 mol/L phosphate buffer, pH
6.8; 0.2 mL/100 g body) by gavage once a week for the initial 8 wk of
the experiment. One hour before termination (1300–1600 h),
5-bromo-2'-deoxyuridine (BrdU; Sigma Chemical, St. Louis, MO) was
injected into the peritoneal cavity (20 mg of BrdU dissolved in 0.1 mL
dimethyl sulfoxide and 0.9 mL saline; 0.5 mL/100 g body). Major organs
were immediately excised and weighed.
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At the end of the experiment, the colon was removed, slit open
longitudinally from cecum to anus, placed on a paper towel and fixed in
10% neutral formalin for 24 h. The colon was embedded in paraffin
and examined histologically after staining with hematoxylin and eosin.
Tumors in the colon were classified into two types, i.e., adenomas and
adenocarcinomas. Adenomas were tumors with no evidence of invasion of
muscularis mucosa. Adenocarcinomas were tumors with evidence of
invasion of muscularis mucosa. Tumor size was measured by image
analysis (16)
. Immunohistochemical analysis of BrdU
labeling was performed for colonic mucosa. The BrdU staining method was
described elsewhere (16)
. BrdU-positive cells in the
normal colonic mucosal epithelium were counted under the microscope at
a magnification of X200. The labeling index of proliferation cells was
the percentage of positive cells in colonic mucosal epithelium
throughout the entire colon, which was examined by observing at least
40 crypts. Immunohistochemical analysis of c-myc and
c-fos proteins was performed for the normal colonic
mucosa. The c-myc and c-fos staining was
as follows. After deparaffinization, rabbit polyclonal
anti-c-myc antibody (Santa Cruz Biochemistry, Santa
Cruz, CA) and rabbit polyclonal anti-c-fos antibody
(Oncogene Research Product, Cambridge, MA) were applied to the
specimens, and they were stained by Vectastain Elite ABC Kit (Vector
Laboratories, Burlingame, CA). The c-myc– and
c–fos-positive cells in the colonic mucosal epithelium
throughout the entire colon were counted under the microscope at a
magnification of X200. For each rat, at least 40 crypts were observed
for the analysis of c-myc– and
c-fos–positive cells.
Fecal analysis.
Feces were dried and ground into a powder. Fecal fat was determined
gravimetrically after lipid extraction by the method of Folch et al.
(17)
. The composition of fecal bile acids was measured by
gas-liquid chromatography according the method described elsewhere
(18)
.
Statistical analysis.
Values are presented as means ± SEM. Data were
analyzed by Student’s t test except for tumor incidence
data, which were analyzed by the 
2 test. Differences
with P < 0.05 were considered significant.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Final body weight (casein, 572 ± 17 g; BWP, 542 ± 12 g) and food intake (casein, 2344 ± 50 g/124 d; BWP, 2244 ± 62 g/124 d) were not affected by dietary manipulation. Perirenal fat pad weight in the BWP group (16.5 ± 1.2 g) was lower than that in the casein group (20.9 ± 1.6 g) (P < 0.05). Weights of tissues including liver, spleen, adrenal, kidney and testis were not affected by dietary manipulation (data not shown).
Incidence of bloody stool and ear duct tumor.
On the final day of the experimental period, nine casein-fed rats
had bloody stools, but none of the BWP-fed rats did (P
< 0.05, Table 2
). BWP intake tended to reduce the incidence of ear duct tumor, which is
often observed when colon cancer is present (casein: 13/20, 65%; BWP:
7/20, 35%) (P = 0.12).
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The incidence of colonic total tumors and adenomas was not
significantly different between the two groups. Also, there was no
difference in the tumor size between the two groups (data not shown).
However, consumption of BWP caused a 47% reduction in the incidence of
colon adenocarcinoma compared with casein (P < 0.05,
Table 2
). The number of adenocarcinomas tended to be reduced by dietary
BWP (P = 0.16).
Cell proliferation.
The rats fed BWP had a significantly lower labeling index of
proliferation cells in the normal-like colonic epithelium than
those fed casein (P < 0.05, Table 3
). The position of the highest BrdU-labeled cell was lower in the
BWP group (P < 0.05). The labeling index of
c-myc and c-fos proteins in the normal-like
colonic crypt was reduced by BWP consumption (P < 0.05) as was the number of cells per crypt column (P < 0.05).
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Fecal wet and dry weights were elevated by BWP intake (P
< 0.05, Table 4
). Fecal concentrations of total lipids and bile acids in the BWP group
were higher than those in the casein group (P < 0.05).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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. This study further demonstrated that dietary BWP
significantly reduced the incidence of colon adenocarcinoma compared
with dietary casein. Additionally, consumption of BWP markedly
suppressed the incidence of bloody stool, possibly relating to tumor
development. The results suggest a strong suppression of colon
carcinogenesis by dietary BWP. In this study, consumption of BWP reduced colonic cell proliferation and protein expression of c-myc and c-fos relating to cell proliferation. The results suggest that reduced cell proliferation is responsible at least in part for the protective effect of BWP against colon carcinogenesis. The position of the highest BrdU labeled cell was also significantly lower in the BWP group, suggesting that consumption of BWP reduced the region in which cell proliferation was occurring. The number of cells per crypt column was slightly reduced by BWP. It is possible that consumption of BWP reduced the number of cells in the colonic mucosa by reducing cell proliferation.
Kayashita et al. (8)
showed a chemopreventive effect
of BWP against the development of
7,12-dimethylbenz[a]anthracene-induced mammary tumor in rats. This
effect of BWP has been considered to be due at least in part to reduced
serum estrogen (8)
. Kato et al. (3)
observed
a reduction in gastrointestinal transit time of the contents from
stomach to anus in rats fed BWP compared with casein because of lower
digestibility of BWP. Thus, the physiologic functions of BWP appear to
be similar to those of some dietary fibers and resistant protein
(3
,5)
. Some dietary fibers reduce fecal concentrations of
total lipids and bile acids (19
20
21
22)
. Because the
digestibility of BWP is relatively low (apparent protein digestibility
in rats fed the diets containing 200 g protein/kg diet: casein,
95.6%; BWP, 76.7%) (5)
, it may be indigestible by the
digestive enzymes in the small intestine, and its indigestible fraction
may dilute the lipids and bile acids in the content of large intestine,
leading to suppression of colon carcinogenesis. To test this
possibility, we examined the fecal lipids and bile acids. However, we
did not observe that BWP intake reduces fecal concentrations of total
lipids and bile acids, suggesting that the preventive effect of BWP is
mediated by a mechanism not involving a lower concentration of total
lipids and bile acids in the content of large intestine. Some
indigestible proteins, namely resistant protein, can alter the large
bowel microflora and result in the production of SCFA that have
potentially beneficial effects on large bowel physiology
(13
,23)
. It is necessary to examine whether BWP suppresses
colon carcinogenesis by altering SCFA in large intestine.
BWP contains 2.8-fold more arginine than casein (4)
.
The content of arginine in the BWP diet is equivalent to the content of
the casein diet supplemented with 15 g/kg arginine. It has been
reported that consumption of arginine (in a 10 g/L
arginine-containing drinking water) inhibits the development of
colon tumors in rats fed DMH by reducing cell proliferation
(24
,25)
. This protective effect of arginine has been
considered to be due to reduced cell proliferation and nonspecific
immune enhancement of the tumor-bearing host (24
,25)
.
Recently, supplementation of 50 g/kg glycine to a 150 g/kg casein diet
was reported to inhibit the growth of B16 melanoma tumors in mice
(26)
. BWP contains 2.5-fold more glycine than casein
(4)
. Although the apparent digestibility of BWP is 
20%
lower than that of casein (5)
, the availability of
arginine and glycine from BWP appears to be higher than that from
casein because of much higher amounts of these amino acids in BWP
(4)
. Thus, arginine and glycine might be the components of
BWP responsible for its preventive effect. BWP contains other minor
components such as lipids and fiber. (5)
. Thus, it is
necessary to examine whether these components are involved in the
protective effect of BWP against colon carcinogenesis.
In conclusion, the present study demonstrated a chemopreventive effect of BWP against colon carcinogenesis. The preventive effect of BWP appeared to be associated with reduced cell proliferation. Because our study was conducted with rats that were fed DMH for the initial 8 wk, the possibility that BWP affects the initiation stage of colon carcinogenesis remains to be studied.
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FOOTNOTES |
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Manuscript received December 11, 2000. Revision accepted March 23, 2001.
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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