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 These consist of increased mineralization of bones via action of genistein (Piastowska et al., unpublished), distorted insulin secretion - agglutinins (though no changes in plasma glucose were observed) (4, 5), and impaired digestion induced by enzyme inhibitors (6)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 al. 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
Animal preparation

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 libitum. 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.

Table 1. Composition of the diets.

1Min. Mix- mineral mixture AIN-93G (MP Biomedicals, Inc.): CaHPO4*2H2O, K2HPO4, K2SO4, NaCl, CaCO3, Na2HPO4*12H2O, MgO, C3H4(OH)(COO)3Fe*3H2O, Zn(CH3COO)2*2H2O, MnCO3, Cu(CH3COO)2*H2O, KJ, citric acid 2Vit. Mix- vitamin mixture AIN-93VX (MP Biomedicals, Inc.): vit. A, D3, E, choline, PABA, inositol, niacin, Ca pantothenate, vit. B1, B2, B6, B12, folic acid, biotin

Staining and analysis

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.

Statistical evaluation

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 P0.05.


RESULTS
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. 1 B). 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. 1 C). 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).

Fig. 1. 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.

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.

Influence of raw and boiled soybean on enterocyte turnover in mid jejunum mucosa

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).

Fig. 3. 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).

DISCUSSION
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. (19).

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 and B). 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 and D) 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.

Acknowledgements: 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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