The content and the quality of proteins in
soybean make it the perfect source of dietary supplementation for both human
and animals. Furthermore, soybean extracts are widely used as a replacement
for animal protein by vegetarians and in infants with cow milk allergies and
lactose intolerance (1). Unfortunately high content of bioactive compounds present
in soybean makes it a tricky diet supplement. Among most potent anti-nutritional
substances are enzyme inhibitors, polyphenols (e.g. phytoestrogens), goitrogens,
phytates, saponins, sugars (e.g. stachyose) and agglutinins (lectins) (2). The
biological effects of some of them are easily abolished by thermal treatment,
but the activity of others remains. Heating of the soybean gets partially rid
of enzyme inhibitors and some polyphenols (3). The biological effects of soybean
are versatile, and can be observed on different levels. The effects related
to the soybean anti-nutritional and hormone-modulating properties characterize
influence at the level of the whole organism. These consist of increased mineralization
of bones via action of genistein (Piastowska et al
., unpublished), distorted
insulin secretion - agglutinins (though no changes in plasma glucose were observed)
(4, 5), and impaired digestion induced by enzyme inhibitors (6) and stachyose
(7). On the other hand, changes in organ and tissue ultrastructure depend mostly
on the agglutinins (4, 5) and polyphenols (8). These changes were observed mostly
in the gastrointestinal tract and related organs. Agglutinins were reported
to decrease the overall body weight (5) while overgrowth of small intestine
and pancreas (4) were observed due to stimulation by cholecystokinin. Chen et
. reported that genistein, in concentrations similar to those present
in infant milk formulas, induced cell cycle arrest and significantly decreased
the mitotic index in piglets, though without changes in small intestine architecture
(9) and showed that genistein inhibits Caco-2 cell proliferation in vitro
(10). Similar results were reported by Mekbungwan et al
. (11) who found
soy-induced changes in villi shape and small intestine digestibility, but not
in organ length and mass in piglets. Moreover Drackley et al
. (12) showed
that soy protein supplementation to milk formula reduced growth and caused severe
alterations in the small intestinal morphology in calves. Villus height and
crypt depth remained unchanged in the duodenum, but were severely decreased
in the jejunum. In ileum the crypt depth remained unchanged, while villus height
decreased. Soy protein influence was so strong that even addition of L-glutamine,
a potent growth promoter, was not enough to reduce the negative effects (12).
In Atlantic salmon, supplementation with soy proteins lead to a mild decrease
in enterocyte proliferation in the mid intestine, while in the distal part cell
proliferation was significantly higher (13). In the large intestine soybean
does not alter the enterocyte turnover (14), positively contributes to microflora
growth and composition (15, 16), and was reported to have at least some anti-tumor
potential, as it alters the global gene expression in colon during tumorigenesis
in rats (17). Unfortunately studies on mechanisms behind observed changes until
recently were not undertaken. In this article we focused on the soy-induced
changes in the enterocyte turnover in growing rats. There were no reports on
the intestine mucosa turnover in small intestine in rats available in literature.
In the preliminary study we quantitatively evaluated the mitotic, programmed
cell death (PCD) apoptotic and autophagy indexes, and the extent of DNA alterations
in healthy small intestine mucosa of control rats. According to our findings
and previous reports (Biernat et al
., unpublished, 12) on piglets and
calves, we designated the middle part of the jejunum for the analysis of soy-induced
modifications of studied processes. This part was found the most suitable due
to relatively small variability in the gut lumen associated with temporary fluctuations
in digestive secretions, pH, water-electrolyte balance and nutrients flow.
MATERIAL AND METHODS
Experiment was conducted on eight-week old Wistar rats, all experimental procedures
were approved by the local ethical committee. Rats were divided into three experimental
groups: 1) control group fed with a standard rat chow: CTRL (n=10); 2) sample
group fed with a standard rat chow supplemented with raw soybean: RS (n=10);
3) sample group fed with a standard rat chow supplemented with boiled soybean:
BS (n=10). Composition of the diets is presented in Tab. 1
. Rats were
maintained in standard conditions (12:12 h light:dark cycle, 24°C) and fed ad
. Soybean was soaked for 24 h in room temperature water and afterwards
was boiled for 15 min. Soybean supplementation was adequate to daily intake
of human adolescent (27.5 g per day). After experiment, rats were euthanized
and intestine samples were collected from the middle of duodenum (DUO), proximal
(PROX), mid (MID) and distal (DIST) jejunum, respectively, 25, 50 and 75% of
length from Treitz ligament, and ileum (ILE). Tissue samples were embedded in
a freezing medium, then frozen in the liquid nitrogen and stored at -80°C.
Staining and analysis
Composition of the diets.
Mix- mineral mixture AIN-93G (MP Biomedicals, Inc.): CaHPO4*2H2O,
NaCl, CaCO3, Na2HPO4*12H2O,
KJ, citric acid
2Vit. Mix- vitamin mixture AIN-93VX (MP
Biomedicals, Inc.): vit. A, D3, E, choline,
PABA, inositol, niacin, Ca pantothenate, vit. B1,
B12, folic acid, biotin
Slices of intestine samples (10 µm) were rinsed with PBS and labeled with the specific sets of antibodies:
- mitosis: anti-Ki-67-FITC-conjugated antibodies (BD Pharmingen, San Diego, CA, USA). The Ki-67 in an absolute requirement for cell progression through the division cycle as it organizes chromatin structure. It is expressed in every phase of cell-division cycle except G0 phase thus allowing quantification of whole population of dividing cells;
- apoptosis: anti-Cpp32 fragment of active caspase 3 (DAKO, Glostrup, Denmark), secondary swine anti-rabbit FITC-conjugated anibodies (DAKO, Glostrup, Denmark). Active caspase 3 is a major effectory caspase involved in execution, irreversible phase of apoptosis;
- autophagy: anti-MAP I LC3 (Santa Cruz Biotechnology, Santa Cruz, CA, USA), secondary antibodies - Alexa Fluor 488 (Molecular Probes, Eugene, OR, USA). MAP I LC3, the only reliable marker of PCD II, occurs on the circumference of nucleus during amino-acids starvation and is involved in formation of autophagosome membranes;
- DNA damage: anti-p53-FITC-conjugated antibodies (DAKO, Glostrup, Denmark). The p53 protein is expressed in the case of DNA damage and facilitates "cell cycle arrest" in a G1 phase allowing either the repair of DNA or elimination of the damaged cell.
Afterwards cell nuclei were counterstained with 7 aminoactinomycin D (7AAD) (Sigma-Aldrich Corporation, St. Louis, MR, USA). Images were acquired with the use of FV-500 laser scanning confocal microscope (Olympus Polska sp. z o.o., Warsaw, Poland), and quantitatively analyzed using the Microimage - image analysis software (Olympus Polska sp. z o.o., Warsaw, Poland). At least 14 images were analyzed for each data point.
The data were statistically analyzed with the use of the GLM model in the SPSS
statistical package software (SPSS, Chicago, IL, USA). Scheffe test was performed
to find significant differences between the means at P
Enterocyte turnover in healthy gut
Evaluated indexes are presented on Fig. 1
. Mitotic indexes evaluated
in subsequent parts of small intestine in rats on the basis of the Ki-67 expression
were 7.3 ± 1.7%, 13.6 ± 1.5%, 11.8 ± 2.9%, 5.2 ± 0.8% and 8.7 ± 1.4% for duodenum
(DUO), proximal jejunum (PROX), mid jejunum (MID), distal jejunum (DIST) and
ileum (ILE), respectively (Fig. 1 A
). Apoptotic indexes did not differ
significantly and varied from 12.3 ± 2.6% (ILE) to 27.1 ± 3.7% (DUO) (Fig.
). Evaluated indexes of autophagy were 1.8 ± 0.2%, 2.2 ± 0.4%, 1.5 ±
0.2%, 1.3 ± 0.1% and 1.1 ± 0.2% in subsequent segments of small intestine (Fig.
). DNA damage measured as a percent of the p53-positive enterocytes was
the lowest in PROX (4.4 ± 0.6%) and the highest value did not exceed 8.7 ± 1.5%
in DIST jejunum (Fig. 1 D
). Statistically significant changes were found
between PROX and DIST in mitotic index (P=0.02) and interestingly, the autophagy
index between the same segments tended to differ (P=0.052). Furthermore packets
of neighboring cells undergoing apoptosis were observed in various small intestine
segments (Fig. 2 - arrow
Enterocyte turnover in the small intestine of eight-weeks old rats from
the control group. Mitotic (A), apoptotic (B), autophagy (C) and DNA damage
indexes (D) presented as geometric mean ± SEM. Statistical details presented
in appropriate section of results.
Influence of raw and boiled soybean on enterocyte turnover in mid jejunum mucosa
|| Fig. 2. Typical packet of several apoptotic cells (arrow) observed on the villi of control eight-week old rats. Active caspase 3 expression marked with FITC (green), DNA counterstained with 7AAD.
The influence of raw (RS) and boiled (BS) soybean supplementation was evaluated
in the mid jejunum only (Fig. 3
). Mitotic indexes were 11.8 ± 2.9%, 13.4
± 1.2% and 25.6 ± 4.1% in CTRL, RS and BS groups, respectively (Fig. 3 A
The significant changes were found between CTRL and BS (P=0.026). In the apoptotic
index, no significant changes were found and indexes varied from 13.1 ± 1.5%
in CTRL rats to 21.2 ± 6.8% in BS rats (Fig. 3 B
). Although autophagy
was strongly pronounced in the RS group, no significant differences were found,
and the index did not exceed 6.6 ± 2.1% (Fig. 3 C
). Indexes evaluated
for the p53-positive enterocytes were 7.9 ± 1.5%, 13.6 ± 1.9% and 12.2 ± 2.8%,
respectively, for CTRL, RS and BS groups (Fig. 3 D
). Significant differences
were found between CTRL and RS groups (P=0.039).
The effect of raw (RS) and boiled (BS) soybean supplementation in the
diet on the mitotic (A), apoptotic (B), autophagy (C) and DNA damage (D)
indexes in the mid jejunum mucosa of eight-week old rats. Bars represent
geometric mean ± SEM, different letters over the bars indicate statistical
significance versus control group (CTRL), p0.05
(ANOVA followed by Scheffe test).
Up to date, no description on rat enterocyte turnover was found in the literature.
Our investigation showed for the first time the extent of major processes contributing
to enterocyte turnover in the small intestine of young rats. Cell turnover was
evaluated on the basis of mitotic, apoptotic and autophagy (two major forms
of programmed cell death), and p53 indexes. The p53 index was used as the marker
of DNA damage, as the p53 protein recognizes DNA alterations and stops cell
in G1 arrest for either DNA repair or apoptosis. Analyzes established mean mitotic
index at 9.3 ± 1.69%, and the significant (P=0.020) difference between the proximal
and distal jejunum was found. In apoptotic and p53 indexes no significant changes
were found, with mean values oscillating around 19.7 ± 3.4% and 6.6 ± 0.6%,
respectively. Autophagy index did not vary significantly, being in the range
of 1.1 ± 0.2% (ILE) and 2.2 ± 0.4% (PROX). It is interesting that changes between
the proximal and distal jejunum were on the verge of significance (P=0.052),
though no correlation was found between mitosis and autophagy (Fig. 1
It is worth mentioning that the pattern of cell death (packets of neighboring
cells simultaneously undergoing apoptosis, Fig. 2
) in the young rat enterocytes
was similar to that previously reported in the newborn piglets by Biernat et
al. (18) and Godlewski et al
High content and quality of soybean proteins was utilized as a significant source
of diet in human and animals alike. Unfortunately the high content of bioactive
substances present in soy makes it the tricky diet supplement (2). The negative
effect of dietary soybean on intestinal mucosa was reported by various authors
(2, 4, 5, 8, 9, 11), and usually was associated with the raw soybean. The protective
role of thermal processing of soybean is widely considered as a way to lessen
the impact of anti-nutritional bioactive compounds on small intestine (3). Our
studies showed that reduction of negative effects of heated soybean observed
by various authors are related to increased mitosis rather than to decreased
apoptosis ratio. The significant increase in mitotic ratio (p=0.026) synchronized
with a tendency toward an increase in apoptosis ratio, was observed in the boiled
soybean-fed group (Fig. 3 A
). On the contrary in the raw
soybean-fed group, the mitotic index was on the level stated in the control,
meanwhile the apoptotic index was elevated displaying the intestine mucosa alterations
found previously (4, 5, 8, 9, 11). These findings were also confirmed by correlation
analysis. The overall (n=20) correlation coefficient between the mitosis and
apoptosis indexes was r=0.59 at P=0.006, while the very same coefficient calculated
within the group fed raw soybean (n=9) was not significant (r=0.08; P=0.830).
The highest values of autophagy and DNA damage indexes were observed in rats
fed raw soybean (Fig. 3 C
) with the p53 index significantly
different from the one found in control group (p=0.039). The large data scatter
in BS group, however, did not allow to draw ultimate conclusions but the index
of DNA damage was almost on the level estimated in the RS group.
To conclude, present data demonstrate that modification of soybean by soaking and subsequent boiling markedly influences the enterocyte turnover in the small intestine mucosa. Increased mitotic ratio in the gut of rats fed with boiled soybean masks the negative effects of soybean on the small intestine structure.
Supported by scientific grant from National Committee for Scientific Research,
Poland No: PBZ-KBN-093/P06/2003 and university grant No: 504 - 02310015.
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