The Effect of Soybean Trypsin Inhibitor on the Generation of Oxygen Free Radical in Pancreas of Mice during Different Growth Periods

Abstract

The present study was undertaken to evaluate the effect of soybean trypsin inhibitor (STI) on the generation of oxygen free radical in pancreas of mice during different growth periods. Mice were divided into three groups and fed with different diets for 1, 2, 3, 4 and 5 weeks separately. The results showed that, MDA content of STI group was increased in the first two weeks, and its maximum appeared in the 3rd week, then it was dropped. Glutathione (GSH) content in serum and pancreas and total anti-oxidative capacity (T-AOC) in serum were dropped in the first two weeks, their minimum appeared in the 3rd week, then they were increased. However, the change of glutathione peroxidase (GSH-Px) in serum and pancreas and T-AOC in pancreas showed opposite trend. The present study indicates that the effect of STI on free radicals level in vivo of mice is relative to growth periods.

Introduction

As all know, soybean is not only an important source of vegetable protein but also has a better balance of amino acid. So its protein has been widely used in human diets, such as soy sauce, soy milk, tofu, flour, etc. (Leibovitz & Siegel, 1980). However, because soybean contains anti-nutritional factors which inhibit the growth of animals and hinder digestion, absorption and utilization of nutrients (Baintner, 1981; Herkelman & Cromwell, 1990; Nitsan, 1986; Li et al., 2000; Naim et al., 1982), its application in human food and animal feeding is severely limited. Therefore, many people begin to carry out scientific research on soybean anti-nutritional factors, which has been become the focus of the domestic and international human and animal nutrition. Research on this area has very important significance to improve food safety and ensure food nutrition. Studies have found that soybean contains more than ten kinds of anti-nutritional factors (Jin & Tian, 2005). Soybean trypsin inhibitor (STI) is one of the main anti-nutritional factors in raw soybean, belongs to polypeptide or protein and consists of 72–197 amino acid residues (Yang et al., 2004). 40% of the anti-nutritional effect caused by soybean is attributed to it. In addition, it can also lead to some physiological responses such as pancreatic hypertrophy and hyperplasia and even pancreatic tumor (Li, 2003; Corring & Chayvialle, 1987), but their mechanisms are still not elucidated at present.

With continuous development of scientific research, people become to realize the close relationship between free radicals and diseases. Under normal circumstances, the generation and elimination of free radicals maintains a physiological equilibrium state. Once this balance is destroyed, the excessive free radicals will have a direct effect on cells, cause cell damaged, and thus trigger a variety of diseases (Hare & Stamler, 2005; Berry & Hare, 2004; Dalle-Donne et al.). After the animals ingested diets containing STI, trypsin and chymotrypsin in the intestinal tract combined with trypsin inhibitor (TI) and formed the enzyme-inhibitor complexes. These complexes could be discharged through feces and resulted in the decrease of enzymes quantities in the intestinal tract, which caused pancreas to compensatory reaction, that is, pancreas was stimulated to secrete and synthesize more trypsin supplied to the intestinal tract (Gallaher & Schneeman, 1986). As is known to us all, during the synthesize process of DNA, mRNA and enzyme, ATP is needed to synthesize purine and pyrimidine and activate amino acid. The generation of ATP is accompanied with the production of free radicals.

In addition, our former findings suggested that oxidative stress may occur in digestive organs after mice ingest a raw soybean diet, which was attributed to antinutritional factors in raw soybean that increased free radicals levels (Gu et al., 2011). Therefore, according to the above theory knowledge and literature report, we speculated that the level of oxygen free radicals may be increased by a dose of STI. This present study is designed to investigate the effect of STI on the generation of oxygen free radical in pancreas of mice during different growth periods. Meanwhile, vitamin C (VC) was supplemented to diet in order to further study the change trend of free radicals under the antioxidant intervention.

Materials and Methods

Animals and Diets    180 male KM mice (body weight, 18 ± 2 g) were used in this study. All animals were housed under a controlled atmosphere (temperature, 23°C ± 1; relative humidity, 55 ± 5%; and a fixed 12 h light: dark cycle, light 0700 to 1900 h). Prior to the feeding experiment, they were allowed free access to deionized water and a control diet for 1 week to allow acclimatization to these conditions. Then all animals were randomly divided into three groups, each group consisting of 60 mice. Control group received a control diet; STI group received a treatment diet, that is, control diet containing 200 mg STI per 100 g diet; STI + VC group received treatment diet supplemented with 1500 mg/kg vitamin C, that is, control diet containing 200 mg/100 g STI and 1500 mg/kg vitamin C. All animals were fed for 1, 2, 3, 4 and 5 w separately. Soybean trypsin inhibitor was provided by our laboratory and its specific activity was 4600 U/mg. The composition of the control diet is shown in Table 1. All mice were allowed free access to the experimental diets and deionized water throughout the experimental period. The care and use of the mice followed the institutional guideline of Jilin Agricultural University.

Table 1. Composition of the diets (g/kg)^a

	Ingredient	Diet
Protein	Caseinb	200
Carbohydrates	Corn starchb	660
Fat	Soybean oilc	50
Fibers	Cellulose powderb	30
Others	Mineral mixtured	50
	Vitamin mixturee	10

Note:

^a The diets were semipurified, isoenergetic (16.33 MJ/kg).

^b Changchun, China.

^c The commercial product (50 g/kg) provides 11.81% of energy. The soybean oil provides the following fatty acids: 14:0, traces; C16:0, 10.3; C16:1 ω-7, 0.1; C18:0, 3.9; C18:1 ω-7 + ω-9, 22.1; C18:2 ω-6, 54.8; C18:3 ω-3, 7.5; C20:0, 0.4; C20:1 ω-9 + ω-11, 0.2; C22:0, 0.4; C22:5 ω-3, traces; C24:0, traces; sum of saturated fatty acids (S), 15; sum of monounsaturated, 22.4; sum of polyunsaturated fatty acids (P), 84.7; P/S, 5.65; Σω-6/Σω-3, 7.3.

^d The salt mixture provides the following amounts (g/kg diet^ȡ1): Ca, 4; K, 2.4; Na, 1.6; Mg, 0.4; Fe, 0.12; trace elements: Mn, 0.032; Cu, 0.005; Zn, 0.018; Co, 0.00004; I, 0.00002.

^e The vitamin mixture provides the following amounts (mg/kg diet^ȡ1): retinol, 12; cholecalciferol, 0.125; thiamin, 40; riboflavin, 30; pantothenic acid, 140; pyridoxine, 20; inositol, 300; cyanocobalamine, 0.1; ascorbic acid, 1600; (dL) α-tocopherol, 340; menadione, 80; nicotinic acid, 200; paraaminobenzoic acid, 100; folic acid, 10; biotin, 0.6; choline, 2720.

Preparation of STI    In this experiment, the raw material was defatted soybean meal. The crude extract of the soybean trypsin inhibitor was obtained by the extraction of phosphoric acid buffer (pH7.6), thermal denaturation(65°C) and ammonium sulfate precipitation. After purified by DE-52 ion exchange, affinity chromatography and sephadex G-75 gel filtration, STI with purification factor of 73.85 was obtained.

Sampling Procedures    At the end of the experimental period, mice were deprived of food overnight but had free access to deionized water. Mice were sacrificed by decapitation. Blood samples were harvested and centrifuged at 4000 × g for 3 min at 4°C and then the serum was separated, kept at −20°C until the oxidative and antioxidative parameters levels were measured. Pancreas was removed and homogenized immediately with DY 89-II homogenizer (Ning Bo Scientz Biotechnology Co., Ltd.) fitted with Teflon plunger, in ice chilled 10% KCl solution (10 mL/g of tissue). The suspension was centrifuged at 671 × g at 4°C for 10 min and clear supernatant was used for the following estimations of activities of GSH-Px, GSH, T-AOC and level of MDA by spectrophotometric methods. The protein content was determined using the method of Lowry et al. (Lowry et al., 1951).

Oxidative Parameter Determination    Level of lipid peroxidation was measured using TBA reaction by Koca et al's method (Koca et al., 2010). This method was used to obtain a spectrophotometric measurement of the color produced during the reaction to TBA with MDA at 532 nm.

Antioxidative Parameters Determination    T-AOC was determined according to Koc et al's method (Koc et al., 2011). Briefly, potent free radical reactions were initiated with the production of a hydroxyl radical via Fenton reaction and rate of reactions was monitored by following the absorbance of colored dianisidyl radicals. Using this method, antioxidative effect of the sample against potent free radical reactions was measured at 550 nm. The non-enzymic antioxidant reduced GSH was analyzed by the method of Ma Ming et al. (Ma et al., 2009). 1.0 mL of homogenate was precipitated with 1.0 mL of sodium phosphateethylenediaminetetraacetic acid (EDTA) buffer and the precipitate was removed by centrifugation. To 0.5 mL of supernatant, 2.0 mL of 5,5Ⅎ-dithiobis (2-nitrobenzoic acid) (DTNB) was added and the total volume was reached to 3.0 mL with phosphate buffer. The colour developed was read at 412 nm. GSH-Px activity was measured according to the method of Sabuncu et al. (Sabuncu et al., 2001). One unit of GSH-Px was defined as a decrease in the log of mmol GSH per minute and was expressed in unit per mg protein. The automatic decrease of GSH without enzyme (control reaction under same condition) was subtracted from the calculation.

Statistical Analysis    Processing of the results was performed using the SPSS 18.0. Data are reported as means ± SD, n = 10. Differences between mean values were determined by ANOVA. Multiple comparisons between the groups were performed using Tukey method. Differences with p < 0.05 were considered significant.

Results

Viscera indexes of mice    As shown in Table 2, the viscera indexes of mice at 5 weeks, as a function of body weight, the pancreas to body weight ratio of STI group showed a significant increase (p < 0.05) compared with that of control group. In absolute terms, liver, spleen, stomach and duodenum to body weight ratio of the STI group were slightly smaller than those of control group. Kidney to body weight ratio was slightly larger than that of control group.

Table 2 Relative tissue weight of mice (g/100 g body weight)

	Control Group	STI Group	STI+VC Group
Pancreas	0.09 ± 0.02	0.12 ± 0.02*	0.11 ± 0.01
Liver	4.76 ± 0.09	4.66 ± 0.36	4.85 ± 0.31
Spleen	0.59 ± 0.12	0.39 ± 0.06	0.41 ± 0.09
Kidney	1.45 ± 0.07	1.46 ± 0.10	1.44 ± 0.09
Stomach	0.94 ± 0.16	0.82 ± 0.07	0. 90 ± 0.15
Duodenum	0.42 ± 0.04	0.45 ± 0.08	0.52 ± 0.09

Note: The same point in time, compared with that of control group, values with ^* mean significant difference (p < 0.05); otherwise mean no significant difference (p > 0.05)

Daily Gain    As shown in Fig.1, the body weight of mice in STI group showed a significant decrease (p < 0.05) compared with that of control group at 5th week. Body weight of mice in STI + VC group was higher than that of STI group, and lower than that of control group.

Fig. 1.

Change in body weight. Body weight of mice fed normal diet (Control group), normal diet containing STI (STI group) and normal diet with STI + vitamin C (STI + VC group) for 1, 2, 3, 4 and 5 weeks. Columns indicate mean body weight of control group, STI group, STI + VC group respectively. Data are expressed as means ± SD. Column data without same letter mean significant difference (p < 0.05); with same letter mean no significant difference (p > 0.05).

Oxidative Parameter in Serum and Pancreas of Mice    Fig.2A and Fig.2B showed that, in serum and pancreas, MDA contents in STI group and STI + VC group were increased firstly and then decreased, and their maximum appeared in the third week. Mice fed STI and STI + VC showed significant increase (p < 0.05) in serum MDA content when compared with the control animals in first four weeks; Administration of vitamin C caused a significant decrease (p < 0.05) of MDA content in serum in comparison with those mice in STI group except the 2nd and 5th week. In pancreas , there appeared to be a significant increase (p < 0.05) in MDA content in the STI group and STI + VC group in comparison with those of the control mice except the 5th week; MDA content of STI + VC group were significantly decreased (p < 0.05) when compared with those in STI group in the 3rd, 4th and 5th week.

Fig. 2.

Changes in MDA content in serum (A) and pancreas (B). MDA content of mice fed normal diet (Control group; closed diamond), normal diet containing STI (STI group; closed triangle) and normal diet with STI + vitamin C (STI + VC group; closed square) for 1, 2, 3, 4 and 5 weeks. Columns indicate mean MDA content of control group, STI group, STI + VC group respectively. Data are expressed as means ± SD. Column data marked with different letter mean significant difference (p < 0.05); with same letter mean no significant difference (p > 0.05).

Antioxidant Parameters in Serum and Pancreas of Mice    As shown in Fig.3A and Fig.3B, T-AOC of control group in serum was increased and reached its maximum in the 5th week. But T-AOC of STI group and STI + VC group was decreased firstly and then increased and in the 3rd week reached a minimum. Mice fed STI and STI + VC showed significant decrease (p < 0.05) in serum T-AOC when compared with the control animals during whole periods; Administration of vitamin C caused a significant increase (p < 0.05) of T-AOC in serum in comparison with those mice in STI group except the 2nd week. T-AOC of three groups in pancreas increased firstly and then decreased and all reached a maximum in the 3rd week. There appeared to be significant decrease (p < 0.05) in T-AOC in pancreas in the STI group and STI + VC group in comparison with those of the control mice except the 1st week; T-AOC in pancreas of STI + VC group was significantly increased (p < 0.05) when compared with those in STI group during the first four weeks.

Fig. 3.

Changes in T-AOC activity in serum (A) and pancreas (B). T-AOC activity of mice fed normal diet (Control group; closed diamond), normal diet containing STI (STI group; closed triangle) and normal diet with STI + vitamin C (STI + VC group; closed square) for 1, 2, 3, 4 and 5 weeks. Columns indicate mean T-AOC activity of control group, STI group, STI + VC group respectively. Data are expressed as means ± SD. Column data marked with different letter mean significant difference (p < 0.05); with same letter mean no significant difference (p > 0.05).

Fig.4A and Fig.4B showed that, with time increasing, GSH-Px activities of three groups in both serum and pancreas were increased firstly and then decreased and in 3rd week reached a maximum. Mice fed STI and STI + VC showed significant decrease (p < 0.05) in GSH-Px activity in serum and pancreas when compared with the control animals during whole periods, whereas the activity of GSH-Px in STI + VC group was significant higher (p < 0.05) than that in STI group except the 3rd week (In the 3rd week, GSH-Px activities tended to decrease, but not to any statistically significant degree between STI group and STI + VC group).

Fig. 4.

Changes in GSH-Px activity in serum (A) and pancreas (B). GSH-Px activity of mice fed normal diet (Control group; closed diamond), normal diet containing STI (STI group; closed triangle) and normal diet with STI + vitamin C (STI + VC group; closed square) for 1, 2, 3, 4 and 5 weeks. Columns indicate mean GSH-Px activity of control group, STI group, STI + VC group respectively. Data are expressed as means ± SD. Column data marked with different letter mean significant difference (p < 0.05); with same letter mean no significant difference (p > 0.05).

Fig.5A and Fig.5B showed that, with time increasing, GSH content of three groups in both serum and pancreas were decreased firstly and then increased and in the third week reached a minimum. Mice fed STI showed a significant decrease (p < 0.05) in GSH content in serum and pancreas when compared with the control animals (In 1st and 2nd week, GSH content tended to decrease, but not to any statistically significant degree). In majority of growth stages, GSH content in serum and pancreas in STI + VC group were significantly increased (p < 0.05) when compared with STI group and STI + VC group.

Fig. 5.

Changes in GSH content in serum (A) and pancreas (B). GSH content of mice fed normal diet (Control group; closed diamond), normal diet containing STI (STI group; closed triangle) and normal diet with STI + vitamin C (STI + VC group; closed square) for 1, 2, 3, 4 and 5 weeks. Columns indicate mean GSH content of control group, STI group, STI + VC group respectively. Data are expressed as means ± SD. Column data marked with different letter mean significant difference (p < 0.05); with same letter mean no significant difference (p > 0.05).

Discussion

Trypsin inhibitors (TIs) are now recognized as a major factor in the toxic effects of raw soybean diets seen in some animal species (Garthoff et al., 2002). For example, in the rat, the pancreatic carcinogenicity of di (2-hydroxypropyl)-nitrosamine and azaserine were potentiated by a diet of full-fat, raw soy flour (McGuinness et al., 1981). Diets containing TI stimulate the exocrine pancreas of many species after short-term ingestion. And this increases the production of pancreatic enzymes and causes pancreatic enlargement and cellular hyperplasia (Garthoff et al., 2002). Rats fed diets containing casein and raw defatted soy flour with a high TI activity (100 – 577 mg TI/100 g diet) in 2-year studies developed progressive pathology of the pancreas. And this included diffuse hypertrophy and hyperplasia followed by neoplastic nodules and acinar adenomas (Garthoff et al., 2002). Further studies found that trypsin inhibitor activity isolated from either soybeans or potatoes produced characteristic pathological effects in rat pancreas that were similar to those caused by raw soy (Gumbmann et al., 1989). Meanwhile, experimental studies have suggested that, pathology change of pancreas were related to the overproduction of free radicals (Zhi, 1992). Furthermore, our results prove that soybean trypsin inhibitor could significantly increase the levels of free radicals in pancreas of mice.

Reactive oxygen species (ROS) is well recognised for playing a dual role as both deleterious and beneficial species. Overproduction of ROS (arising either from mitochondrial electrontransport chain or excessive stimulation of NAD(P)H) results in oxidative stress (Marian et al., 2007), a deleterious process that has been implicated in various pathological conditions involving cardiovascular disease, cancer, neurological disorders, diabetes, ischemia/reperfusion, other diseases and ageing (Baker et al., 2004; Valko et al., 2007; Stadtman, 2004; Dalle-Donne et al., 2005; Gupte & Mumper, 2009; Khandrika et al., 2009). Free radicals react with lipid and cause peroxidative changes that result in enhanced lipid peroxidation (Zwart et al., 1999; Girotti, 1985). Malondialdehyde (MDA) was the end products of lipid peroxidation. In our present study, MDA contents of the STI group in serum and pan creatic were increased significantly compared with those of the control group. This result suggests that STI caused a significant increase in lipid peroxidation degree, which indirectly reflects the levels of free radicals in pancreas of mice were increased. With the increase of feeding time, MDA content in the STI group showed a significant upward and then downward trend, and reached a peak in the third week. This result suggests that impact of STI on the level of free radicals in mice is related with growth cycles. Luo Yi et al. (2007) found that oxidative damage occurred in the liver of the larva fish after 2, 4-DCP treatment for 2, 4, 8, 24 and 72h separately, MDA content in vivo was upward trend. Similar results have been found by Chen Ping et al. (2009). It was reported that oxidative damage induced by bleomycin in the lung of rats, model rats were killed on the 3rd, 7th, 14th and 28th d separately, the result showed that MDA content was increased firstly and then decreased and in the third week reached a maximum (Liu et al., 2004), which is similar to our research. The above research reports show that the extent of body damage caused by oxidative stress is relative to growth cycle.

As the free radical scavenging system, T-AOC, GSH and GSH-Px exist in all oxygen-metabolizing cells, prevent cells from damage by free radicals and provide a repair mechanism for oxidized membrane components (Ma et al., 2009), reflect the capacity of nonenzymatic antioxidant defense system. So this study took the above indicators to measure the variety of antioxidant status in pancreas. As data are summarized in Fig.3, Fig.4 and Fig.5, decreased antioxidant parameters of STI group markedly happened to all tested serum and pancreas. This is due to the increase of oxygen free radicals level caused by STI, in order to maintain oxidation and antioxidant balance, the body consumed a lot of antioxidants, which results in the antioxidant defense system being damaged and weakening the body's antioxidant capacity. According to the data, we found that the trends of T-AOC and other antioxidants in the STI group in different growth stages were inconsistent. The contents of GSH in serum and pancreas and T-AOC in serum was decreased firstly and then increased and in the third week reached a minimum, however, the change of GSH-Px in serum and pancreas and T-AOC in pancreas showed opposite trend. Such findings were similar to those previously reported (Luo et al., 2007; Chen et al., 2009; Liu et al., 2004; Tan et al., 2005). These results could further explain, after oxidative stress occurred, change of the antioxidant capacity may be related to the level of oxygen free radical and the growth cycle of body.

Vitamin C is involved in a number of metabolic processes in the human body, including those that may be important for the optimal functioning of the oxygen energy system (Cholewa et al., 2008). It is a powerful antioxidant, helping prevent cellular damage and impairment of the immune system by neutralizing free radicals directly or indirectly. The results show that, administration of vitamin C caused a significant decrease of oxidative parameter and a significant increase of antioxidant parameters in serum and pancreas. This illustrate that vitamin C can decrease the effect of oxidative stress of soybean trypsin inhibitor, weakened oxidative damage induced by reactive oxygen.

In the following study, we will evaluate the effect of different levels soybean trypsin inhibitor on the oxygen free radical levels in pancreas of mice. To research if soybean trypsin inhibitor have antioxidant activities like trypsin inhibitors in sweet potato and casein phosphopeptides.

Conclusions

In summary, our test indicates that soybean trypsin inhibitor exhibits the harmful effect of inducing oxidative stress by increasing the formation of lipid peroxidation and overall impairing enzymatic and nonenzymatic antioxidant defenses in the soybean trypsin inhibitor diet-fed mice. In addition, change trends of oxidant and antioxidant capacity will be relevant to growth cycle of body. Supplement with vitamin C in soybean trypsin inhibitor diet significantly reduced extent of oxidative stress in serum and pancreas of mice, which further demonstrated that soybean trypsin inhibitor may destroy the balance of oxidant and antioxidant in organism by inducing generation of free radicals.

Acknowledgments    The authors want to thank National Natural Science Foundation of China (NSFC, No.31000769), Postdoctoral Library of Jilin Agricultural University and the China Postdoctoral Science Foundation (Grant, NO. 2012 M520690) for the funding.

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