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General Health and Medical Sciences, Vol(1), No (2), December, 2014. pp.

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Impact of Soy Isoflavones on Aflatoxin-induced Oxidative Stress and


Hepatotoxicity in Rats
Mosaad A. Abdel-Wahhab *
Food Toxicology & Contaminants Dept., National Research Center, Dokki, Cairo, Egypt.

Ezzeldein S. El-Denshary
Toxicology & Pharmacology Dept., Faculty of Pharmacy, Cairo University, Cairo, Egypt.

Aziza A. El-Nekeety
Food Toxicology & Contaminants Dept., National Research Center, Dokki, Cairo, Egypt.

Nabila S. Hassan
Pathology Dept., National Research Center, Dokki, Cairo, Egypt.

Ferial M. Abu-Salem
Food Technology Dept., National Research Center, Dokki, Cairo, Egypt.

Nesma A. Z. Sarhan
National Cancer Institute, Cairo, Egypt.

Bertrand H. Rhin
Faculty of Pharmacy, EA 3452 CITHEFOR, Lorraine University, 54001 Nancy Cedex, France.
*Corresponding author: mosaad_abdelwahhab@yahoo.com

Keywords

Abstract

Aflatoxins
Liver
Oxidative stress
Soybeans
Isoflavones

Liver diseases are amongst the most serious health problems in the world today and their prevention and
treatment options still remain limited despite tremendous advances in modern medicine. The aim of this
study was to evaluate the protective effects of isoflavones in soy against aflatoxins (AFs)-induced
hepatotoxicity and oxidative stress in rats. Forty male Sprague-Dawley rats were divided into four groups
and treated for 8 weeks as follow: the control group, the group fed soy-supplemented diet (20% w/w) and
the groups fed AFs-contaminated diet (1.5 mg /kg diet) alone or in combination with soy. Blood and liver
tissue samples were collected for different analysis. The results indicated that AFs-contaminated diet
resulted in significant changes in serum biochemical parameters and oxidative stress markers accompanied
with severe histological changes in liver. Supplementation with soy isoflavones succeeded to restore the
elevation of liver enzymes activities, improved the serum biochemical parameters, antioxidant enzymes,
decreased lipid peroxidation and improved the histological picture of the liver tissue. It could be concluded
that soy protein enriched isoflavones may be a promising agent against liver toxicity.

1.

Introduction

Aflatoxins (AFs) are secondary fungal metabolites produced by Asperigillus parasiticus and A. flavus that have long been recognized as
significant environmental contaminants in foods [1]. AFs especially aflatoxin B1 (AFB 1) are confirmed as a potential carcinogen and was
classified as group I human carcinogen [2]. The extent of the carcinogenicity of aflatoxin depends on the presence of human health factors
including hepatitis B virus infection, nutritional status, sex and age as well as the amount of AFs exposure.
The metabolism of AFB 1 takes place by mixed-function oxidase system at the liver to AFB1 8, 9-epoxide and other hydroxylated metabolites.
These metabolites bind to DNA, forming covalent adducts [3] which interrupt DNA replication causing chromosomal aberrations [4]. It has well
been documented that AFB1 leads to liver-specific carcinogenicity through induction a guanine (a purine) to thiamine (a pyrimidine) substitution
at codon 249 on the p53 gene [5]. Moreover, Abdel-Wahhab et al. [6] reported that AFs are known to produce membrane damage through
increased lipid peroxidation and the generation of free radicals. Furthermore, AFB1 was found to inhibit CD14- mediated nitric oxide production
in murine peritoneal macrophages [7]. Presently, there is no proven effective systemic chemotherapy for hepatocellular carcinoma [8]. So,
prevention has been considered to be the best strategy in lowering the present prevalence of the disease in view of the limited treatment and
negative prognosis of liver cancer [9]. Several reports have reviewed the potential roles of soy or its isoflavones in decreasing the risk of many
types of cancer [10] [11] [12]. Soy isoflavones have been shown to exhibit antioxidant effects and decrease LDL oxidation in vitro and in vivo
by direct free radical quenching ability, with genistein and daidzein being particularly effective [13] [14] [15]. The aim of this study was to
evaluate the protective effects of isoflavones (daidzin, genistein, genistin, daidzein) in soy against AFs-induced oxidative stress in rats.

2.

Materials and Methods

2.1. Chemicals and kits


Aflatoxins (AFs) standards mixture was purchased from Sigma Chemical Co. (St Louis, MO, USA). Transaminase (ALT and AST) kits were
purchased from Spectrum-diagnostics (Cairo, Egypt). Alpha feto protein (AFP) kit was purchased from Immunospec (CA, USA). Total protein,
albumin, Cholesterol, triglycerides, superoxide dismutase, Glutathione peroxidase (GPX) and Lipid Peroxidation (MDA) kits were purchased
from Biodiagnostic Co. (Giza, Egypt).

Mosaad A. A. Abdel-Wahhab, Ezzeldein S El-Denshary, Aziza A El-Nekeety, Nabila S Hassan, Ferial M Abu-Salem, Nesma A. Z Sarhan, Bertrand H Rhin

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General Health and Medical Sciences Vol(1), No (2), December, 2014.

2.2. Aflatoxins production


AFs were produced by the fermentation of corn with Aspergillus parasiticus NRRL 2999 according to Abdel-Wahhab et al. [4]. The fermented
corn was autoclaved, dried and ground to a powder and AFs content was measured by HPLC [16]. The AFs within corn powder consisted of
42% B 1, 12% B 2, 33% G1, and 13% G2 based on total AFs in the corn powder. The corn powder was incorporated into the basal diet to provide
the desired level of 1.5 mg of AFs/kg diet. The diet containing AFs was analyzed and the presence of parent AFs was confirmed and determined
by HPLC.
2.3. Plant materials
Soy bean (Glycine max) was purchased from the Crop institute, Agricultural Research Center, Giza, Egypt in the season 2013.
2.4. Experimental animals
Mature male Sprague-Dawley rats (3 month old and weighed 100-120 g) were purchased from Animal House Colony, Giza, Egypt and were
maintained on standard lab diet (protein: 160.4; fat: 36.3; fiber: 41 g/kg and metabolizable energy 12.08 MJ) purchased from Meladco Feed Co.
(Aubor City, Cairo, Egypt). Animals were housed in an artificially illuminated and thermally controlled room free from any source of chemical
contamination at the Animal House Lab., National Research Centre, Dokki, Cairo, Egypt. After an acclimatization period of one week, the
animals were divided into four groups (10 rats/group) and housed in filter-top polycarbonate cages. All animals were received humane care in
compliance with the guidelines of the Animal Care and Use Committee of the National Research Center, Dokki, Cairo, Egypt.
2.5. Experimental design
All animals in different treatment groups were maintained on their respective treatment for 8 weeks as follow: group (1), untreated control;
group (2), fed soy supplemented diet (20% w/w); group (3), fed AFs-contaminated diet (1.5 mg /kg diet) and group (4), fed AFs-contaminated
diet supplemented with soy. At the end of the treatment period (i.e. day 56), all animals were being fasted for 12 hrs and blood samples were
collected from the retroorbital venous plexus under diethyl ether anesthesia. Sera were separated using cooling centrifugation and stored at -20
O
C until analysis. The following serum biochemical parameters: ALT and AST, total protein, albumin, cholesterol, triglycerides and alpha
fetoprotein were carried out according to the kits instructions. After blood samples were collected, all animals were killed by cervical dislocation
and sample of liver tissues of each animal was dissected, weighed and homogenized in phosphate buffer (pH 7.4) to give 20% w/v homogenate
[17]. This homogenate was centrifuged at 1700 rpm at 4C for 10 min and the supernatant was stored at -70 C until analysis. Lipid peroxidation
(LP) was estimated by measuring the formed malondialdehyde (MDA) using the spectrophotometric method. The level of lipid peroxidation was
expressed as nmol MDA per gram tissue. Hepatic glutathione peroxidase (GPX) activity was determined by the spectrophotometric method.
Hepatic superoxide dismutase (SOD) activity was assayed spectrophometrically. The activity of hepatic glutathione peroxidase and superoxide
dismutase was expressed as unit/ mg liver protein. All the biochemical parameters were determined according to the kits instructions. Other
samples of the liver from all animals within different treatment groups were excised and fixed in 10% neutral formalin followed by dehydration
in ascending grades of alcohol, clearing in xylene and embedding in paraffin wax. Liver sections (5m thickness) were stained with hematoxylin
and eosin (H & E) for the histological examination [18].
2.6. Statistical analysis
All data were statistically analyzed by analysis of Variance (ANOVA) using the General Linear Model Procedure of the Statistical Analysis
System. The significance of the differences among treatment groups was determined by Waller-Duncan k-ratio [19]. All statements of
significance were based on probability of P 0.05.

3.

Results

The results of the current study revealed that exposure to AFs in the diet resulted in a significant increase in ALT, AST, AFP, triglycerides and
cholesterol level accompanied with a significant decrease in albumin and total protein (Table 1). Animals fed soy-supplemented diet showed a
significant decrease in AFP, cholesterol and triglycerides; however, ALT and AST activities, albumin and total protein levels were comparable
to the control. Supplementation of AFs-contaminated diet with soy resulted in a significant improvement in ALT, AST, total protein, cholesterol,
albumin and triglycerides towards the normal values of the control group.
Table 1. The effect of soy supplementation on different treatments on the biochemical serum parameters in rats fed AFs-contaminated diet
Groups
Parameters
ALT (U/ml)
AST (U/ml)
Albumin (g/dl)
Total protein (g/dl)
Cholesterol (mg/dl)
Triglycerides (mg/dl)
AFP (IU/ml)

Control

Soy

AFs

AFs + Soy

63.43 4.20a
174.14 2.32a
4.23 0.14a
8.87 1.21a
60.43 3.63a
65.32 3.51a
8.25 1.23a

61.34 4.76a
171.12 3.22a
4.14 0.75a
9.53 0.77a
52.34 2.57b
53.52 4.11b
7.22 1.43b

128 17.95b
198.25 7.81b
2.54 0.23b
5.82 0.28b
91.69 1.29c
98.16 2.58c
13.86 0.54c

68.34 2.85a
177.5 4.25a
3.29 0.11a
8.92 0.58a
51.56 3.86b
62.37 2.99a
9.58 1.74b

Within each row, means superscript with different letters are significantly different (P 0.05)

The current results also revealed that AFs-contaminated diet resulted in a severe stress in liver as indicated by the significant increase in MDA
and the significant decrease of GPX and SOD in liver tissue (Table 2). However, animals fed soy-supplemented diet showed a significant
decrease in MDA accompanied with a significant increase in GPX and SOD. Animals fed AFs-contaminated diet supplemented with soy showed
a significant improvement in the oxidative stress marker as well as the antioxidant enzymes activity although these values were still differ
significantly than the control group.
The biochemical analyses were further confirmed by the histological examination of the liver sections. The results indicated that liver tissue in
the control group or those fed soy-supplemented diet showed normal central vein and normal hepatocytes (Fig. 1A, B). While animals fed AFscontaminated diet showed hyperplasia, hypertrophy in bile duct epithelial cells around the congested and thick portal tract (Fig. 1C) and
disorganization of hepatocytes architecture (Fig. 1D). Liver of animals fed AFs-contaminated diet supplemented with soy showed marked
improvement in hepatocytes and the central vein (Fig. 1E) and most of hepatic section (Fig. 1F).

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Impact of Soy Isoflavones on Aflatoxin-induced Oxidative Stress and Hepatotoxicity in Rats


General Health and Medical Sciences Vol(1), No (2), December, 2014.

Table 2. The effect of soy supplementation on oxidative stress markers in liver of rats fed AFs-contaminated diet
Groups
Parameters
GPX
(U/g tissue)
SOD
(U/g tissue)
MDA
(nmol/g tissue)

Control

Soy

AFs

AFs + Soy

2425.32
45.32a
1298.84
52.32a
43.63
3.22a

2654.43
23.14b
2987.63
45.32b
38.74
4.77b

908.90
9.11c
881.78
54.59c
130.89
9.62c

2269.56
324.22e
2971.5
53.7d
63.33
7.35d

Within each row, means superscript with different letters are significantly different (P 0.05)

4.

Discussion

As reported earlier in the literature, AFs induce harmful health effects including carcinogenicity, teratogenecity and immunotoxicity [4] [5] [6].
In various diseases, the steady state of pro-oxidants and antioxidants may be disrupted in favor of the former, leading to oxidative stress, which
in turn may affect all types of biological molecules, including DNA, lipids, proteins, and carbohydrates [6]. Thus, oxidative stress may be
involved in processes of diseases such as those resulted from AFs.

(A) (HX& E X 150)

(B) (HX& E X 400)

(C) (HX & E X 200)

(D) (HX & E X 200)

(E) (HX & E X 200)

(F) (HX & E X 100)

Fig. 1. A photomicrograph in liver section of: (A) A control rat showing central veins, portal tracts and hepatic cords
separated with blood sinusoids. (B) A rat fed soy-supplemented diet showing central vein and hepatocytes separated
with blood sinusoids. (c) A rat fed AFs-contaminated diet showing hyperplasia and hypertrophy in the bile ducts
epithelial cells around the congested and thick portal tract, (D) A rat fed AFs-contaminated diet showing the
disorganization of hepatocytes architecture. (E) A rat fed AFs-contaminated diet supplemented with soy showing
marked improvement in hepatocytes and the central vein (F) A rat fed AFs-contaminated diet supplemented with soy
showing marked improvement in most of hepatic section.

Mosaad A. A. Abdel-Wahhab, Ezzeldein S El-Denshary, Aziza A El-Nekeety, Nabila S Hassan, Ferial M Abu-Salem, Nesma A. Z Sarhan, Bertrand H Rhin

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General Health and Medical Sciences Vol(1), No (2), December, 2014.

In the current study, the selected doses of AFs and soy were based on our previous work [4] [20]. No animal mortality was observed in any of
the treatment groups except for two rats died in the group fed AFs-contaminated diet. AFs at 1.5 mg/kg feed caused significant changes in serum
biochemical parameters typical to those reported in the literature [4] [6]. The liver is considered to be the principal target organ for AFs and the
elevated levels of ALT, AST, triglycerides and cholesterol reported herein indicate severe hepatic parenchymal cells injury [6] [21]. Whereas;
the decrease in total protein and albumin indicated liver necrosis and/or kidney dysfunction [22].
AFP is considered a specific biomarker for liver cancer and it is synthesized mainly in the fetal stage; practically no production of this marker
occurs in the normal adult. However, when some adult cells are transformed to cancer cells, the synthesis of AFP commences again. In the
current study, the elevated serum level of AFP in the animals fed AFs-contaminated diet indicated its potent hepatocarcinogens, which enhance
reactive oxygen species (ROS) formation and cause oxidative DNA damage, that may play a role in their carcinogenicity [6]. Therefore, the
current study affirmed that AFs can induce hepatotoxicity and degeneration in liver cells in rats as indicated by the elevation of AFP level in
serum.
MDA, an end product of lipid peroxidation, is widely used as a marker of lipid peroxidation (LP). LP is one of the main manifestations of
oxidative damage and has been found to play an important role in toxicity and carcinogenicity. It is well documented that aflatoxin enhanced
lipid peroxidation that is an indication of free radical mediated toxicity [6]. Free radicals are known to attack the highly unsaturated fatty acids of
the cell membrane and induce LP that is considered a key process in many pathological events induced by oxidative stress. In the present study,
MDA was found to be significantly higher in the animals fed AFs-contaminated diet suggesting a significant effect on LP and supported the
earlier findings [23].
Alteration in the hepatic antioxidant status may be a manifestation of oxidative stress caused by AFs or their metabolites. Both GPX and SOD
are considered enzymatic free-radical scavengers in cells. In the present study, GPX and SOD were found to decline significantly in rats fed AFcontaminated diet. It is well known that SOD plays an important role in the elimination of ROS derived from the peroxidative process in liver
tissues [6]. Moreover, SOD removes superoxide by converting it to H2O2, which can be rapidly converted to water by CAT [24]. Taken together,
the increased level of MDA and the decreased activity of antioxidant enzymes GPX and SOD may be attributed to the free radical formation
which initiates chain reactions of direct and indirect bond formation with cellular molecules (nucleic acids, proteins, lipids and carbohydrates)
impairing crucial cellular processes that may ultimately culminate.
The biochemical results reported herein was further confirmed by the histopathological investigation of the liver tissues. It is clear that animals
fed AFs-contaminated diet showed severe histological changes in liver typical to those reported in the literature for aflatoxcosis. In AFs-treated
group, the liver showed disorganization of hepatocytes architecture, clusters of inflammatory cells and some hepatocytes appeared with
coagulative necrosis or apoptosis. Some sections showed hyperplasia and hypertrophy in the bile ducts epithelial cells around the congested and
thick portal tract and the hepatocytes in the peri-portal zone with coagulative necrosis. These findings are similar to those reported earlier [4].
In a previous work, we reported that soy flour is rich in crude protein which reached 45.8% based on the dry matter. Moreover, the isoflavones
content in 100 g soy recorded 87.4 mg daidzin, 21.4 mg genistin, 47.9 mg daidzein and 10.6 mg genistein [20]. We also reported that isoflavones
genistin and dadzin constitute 99% of the total isoflavones similar to those reported earlier [25] [26]. Soybeans and soy products, which are
relatively enriched in isoflavones, are of particular interest due to the fact that they make up a significant dietary protein source in some areas of
the world [27]. Moreover, soy is rich in phenols which have been reported to exhibit antioxidant activity and have been reported to decrease
LDL oxidation both in vitro and in vivo [13] [28].
Moreover, it is well documented that soybean is composed of macronutriments such as lipids, carbohydrates and proteins. According to a
previous study [26], soybean lipids, which are deprived of cholesterol, contain about 15% of saturated fat, 61% of polyunsaturated fat, and 24%
of monounsaturated fat. Carbohydrates make up about 30% of the seed, with 15% being soluble carbohydrates (sucrose, raffinose, stachyose)
and 15% insoluble carbohydrates (dietary fiber). The protein content of soybean varies from 36% to 46% depending on the variety. These
storage proteins are predominant, such as the 7S globulin (-conglycinin) and 11S globulin (glycinin), which represent about 80% of total
protein content, as well as less abundant storage proteins such as 2S, 9S, and 15S globulins [29] [30].
Soybean also contains micronutriments, which include isoflavones, phytate, soyaponins, phytosterol, vitamins and minerals and soy protein has
long been an important protein source in traditional oriental diets. It shows benefits in decreasing liver lipid accumulation and increasing
antioxidation ability [31] [32]. Previous reports indicated that soy phytochemicals can modulate both phase I and II enzymes in the xenobiotic
metabolizing system [33]. In vivo and in vitro experiments have demonstrated that soy and soy phytochemicals specifically induce quinone
reductase (QR) activity [34].
In the current study, animals fed soy-supplemented diet showed a significant improvement in all the biochemical parameters tested and the
histological picture of the liver. The levels of AST and ALT were lower in the groups fed soy-supplemented diet compared with those received
AFs. Possible explanation for the differential effects of soy on the activities of AST and ALT is that daidzin and daidzein, the main isoflavones
in soybeans, may inhibit the liver damage induced by AFs. On the other hand, supplementation of the diet with soy protein significantly lowered
plasma cholesterol and triglyceride levels caused by AF. Recent reports indicated that soy protein significantly lowered plasma cholesterol
concentrations and body fat accumulation. Soy protein intake also decreased the hepatic lipid depots of triacylglycerols and cholesterol and
decreased the concentrations of lipid peroxides [35]. A possible mechanism of this decrease is the enhancement of bile acid excretion, by which
soy protein acts as dietary fiber to promote bile acid excretion and increase the rate of cholesterol resynthesis in the liver [36].
Previous report has shown that soy protein may improve hyperlipidemia due to its effects on the transcription factors, called sterol regulatory
element binding proteins, which are important in the regulation of enzymes involved in lipid metabolism in vivo and helped prevent the
development of hepatic lipotoxicity [37]. Soy ingestion significantly lowered AFP levels in the serum of rats which may be due to the antiinflammatory properties of soy protein. In this concern, previous studies have indicated that the isoflavones in soy protein may regulate the
inflammatory response and immune function [38]. For example, soy protein was found to regulate bronchial contractions caused by allergens as
a tyrosine kinase inhibitor [39]. Another mechanism for regulating the inflammatory response and immune function may be the decrease of liver
fat and oxidative stress, which indirectly decreases the damage of hepatocytes and lowers AFP level. These results suggest that soy protein may
inhibit the inflammatory response in AFs and further decrease damage to the liver. Moreover, Genistein and Secoisolariciresinol were found to
have anti-cancer properties on MCF-7 and BT20 in vitro and have been suggested as the most potent inhibitor of cancer cell growth [40].
Moreover, the current results revealed that soy supplementation resulted in a significant decrease in LP measured as MDA concentration in liver.
This decrease was accompanied with significant increases in GPX and SOD activities. Consequently, diet supplemented with soy succeeded to
induce a significant improvement in the oxidative stress markers and antioxidant status of the liver tissue in rats treated with AFs. These results
suggested that soy protein may prevent oxidative damage in the liver by lowering plasma free fatty acids and decreasing CYP2E1 expression.
Similar observations were reported recently by Yang et al. [35] who stated that rats fed the soy protein diet showed improved antioxidative
potential due to increases in superoxide dismutase and catalase activities and a decrease in the protein expression of cytochrome P450 2E1.

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Impact of Soy Isoflavones on Aflatoxin-induced Oxidative Stress and Hepatotoxicity in Rats


General Health and Medical Sciences Vol(1), No (2), December, 2014.

5.

Conclusion

The current study revealed that AFs induced sever hepatotoxicity typical to those reported in the literature. Supplementation with soy succeeded
to protect the liver from the toxic effects and oxidative stress. The protective effects of soy may be due to its antioxidant activity, the anti cancer
effect and the enhancement of immune response since it increased the antioxidant capacity of the body and reduced the oxidative stress and
tumor markers. Moreover, soy was found to be contained a combination of active compounds may be more efficacious and safer as chemopreventive agents than individual compounds.

Conflict of Interest
The authors declare that there are no conflicts of interest.

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