Dietary supplementation with whey protein and ginseng extract counteracts oxidative stress and DNA damage in rats fed an aflatoxin-contaminated diet

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Abstract

Aflatoxins (AF) are among the most potent naturally occurring carcinogens and aflatoxin-B1 (AFB1) is classified as a group-1 carcinogen. Since the ingestion of aflatoxins-contaminated food is associated with several liver diseases, the aim of the present study was to evaluate whether AF-induced damage in rats can be counteracted by feeding with whey-protein concentrates (WPC) and Korean ginseng extract (KGE).

Eighty male Sprague-Dawley rats were divided into eight equal groups and treated daily for 30 days as follows: a control group (fed an AF-free diet), a group fed ad libitum an AF-contaminated diet (2.5 mg/kg diet), a group treated orally with WPC (0.5 ml/rat/day), a group treated orally with KGE (20 mg/kg body weight), a group treated orally with WPC + KGE, and three groups that were fed the AF-contaminated diet and were treated orally with WPC, KGE or WPC + KGE, respectively. Throughout the experimental period, animals received WPC or KGE during the consumption of their respective diet. Bone-marrow micronucleus formation, DNA fragmentation, fatty-acid synthesis (FAS) and phospholipid-hydroperoxide-glutathione-peroxidase (PHGPx) mRNA expression, and oxidative stress were assayed in liver and testis. The results indicated that ingestion of aflatoxin resulted in a significant increase in micronucleated normochromatic erythrocytes (Mn-NCE) in bone marrow, DNA fragmentation, FAS mRNA expression and lipid peroxidation in both organs, and a significant decrease in micronucleated polychromatic erythrocytes/micronucleated normochromatic erythrocytes (PCE/NCE) ratio in bone marrow, PHGPx gene expression and GSH in liver and testis. Treatments with WPC and/or KGE had a significant effect on Mn-NCE or the PCE/NCE ratio in bone marrow. However, KGE or KGE + WPC increased PHGPx gene expression and GSH in testis accompanied with a significant decrease in lipid peroxidation in liver and testis and FAS-mRNA expression in liver. WPC, KGE or WPC + KGE treatments combined with exposure to an AF-contaminated diet restored all the test parameters towards control values, although they did not fully reverse the effects of the aflatoxins. It is suggested that the genotoxicity of aflatoxins can be in part prevented by dietary supplementation with WPC, KGE or their combination.

Introduction

Aflatoxins (AF) are potent mycotoxins produced by fungi of the Aspergillus group. They have a wide range of biological activities, such as oxidative stress [1], [2], acute toxicity, teratogenicity, mutagenicity and carcinogenicity [3], [4], [5]. Aflatoxin-B1 is metabolically bio-transformed in the liver into a highly reactive electrophilic compound that interacts with cellular macromolecules [3]. It has been previously reported that drug-metabolizing enzymes (phase-I and phase-II enzymes) and AFB1-adduct formation can be modulated by natural constituents of the diet, nutrients and xenobiotics [6].

Panax ginseng C.A. Meyer is an herbal root that has been used for more than 2000 years throughout countries of the Far-East, including China, Japan, and Korea. Its beneficial effects have been analyzed by extensive preclinical and epidemiological studies [7], [8]. Recently, 20-O-(h-d-glucopyranosyl)-20(S)-protopanaxadiol (IH-901), a novel ginseng saponin metabolite formed from ginsenosides Rb1, Rb2, and Rc was isolated and purified after giving ginseng extract orally to humans and rats [9]. IH-901 has been shown to enhance the efficacy of anticancer drugs in cancer cell lines that were previously resistant to such drugs [10]. IH-901 also exhibits anti-genotoxic and anti-clastogenic activity in rats concurrently treated with benzo(a)pyrene [11], and it induces apoptosis [12]. Moreover, several studies indicated that apoptosis is characterized by DNA fragmentation, chromatin condensation, membrane blebbing, cell shrinkage and disassembly into membrane-enclosed vesicles [13].

Whey-protein concentrates (WPC) are widely used in the food industry as functional and nutritional ingredients due to their wide range of chemical, physical and functional properties. The most important properties of whey proteins are solubility, viscosity, water-holding capacity, gelation, emulsification and foaming [14]. In addition to these general properties, individual whey proteins have their own unique nutritional, functional and biological characteristics. For instance, α-lactalbumin has been claimed as a nutraceutical and a food additive in infant formula owing to its high content of tryptophan [15] and as a protective against ethanol- and stress-induced gastric mucosal injury [16]. β-Lactoglobulin is commonly used to stabilize food emulsions because of its surface-active properties [17]. Bovine serum albumin (BSA) has gelation properties and is of interest in a number of food and therapeutic applications [18], possibly because of its antioxidant properties [19]. WPC is prepared in a special way to preserve the native forms of the cysteine-rich protein in whey (serum albumin, lactoferrin, and α-lactalabumin) which functions as a cysteine donor in cells [20]. It has been shown to represent an effective and safe cysteine donor that serves to replenish GSH during GSH-depletion under conditions of immuno-deficiency [21]. The aim of the present study was to evaluate whether AF-induced damage in rats can be counteracted by feeding with whey-protein concentrates (WPC) and Korean ginseng extract (KGE).

Section snippets

Chemicals

Aflatoxin standards and other chemicals were purchased from Sigma Chemical Co. (St. Louis, MO, USA) if not otherwise mentioned. Chemicals were of the highest purity commercially available.

Whey protein (WPC)

Concentrated whey powder containing 80% protein was purchased from Davisco Foods International, Inc. (Eden Prairie, MN, USA). WPC solutions (15% (w/w), pH 6.9) were prepared by dispersing WPC powder in water. It was administered at a dose of 300 mg/kg bw.

Plant materials

Korean Red ginseng was provided by the Korean Society of

KGE and WPC analysis

The HPLC analyses of the KGE are presented in Table 1. These results showed that individual ginsenoside content in KGE was 0.54, 0.95, 1.02, 0.88, 3.16, 3.72, 1.89, 1.71, 1.32, 4.04, and 0.11 mg/g for Rg1, Re, Rf, Rh1, Rg2, Rb1, Rc, Rb2, Rd, Rg3 and Rh2, respectively. The sum of ginsenosides contents was 19.3 mg/g (1.93%). On the other hand, determination of thiol groups in WPC revealed that it contains 523 μM thiol (SH) groups as cysteine equivalents.

Micronucleus test

The results of the current study indicate that

Discussion

Oxidative damage induced by ROS causes tissue damage by a variety of mechanisms including DNA damage, lipid peroxidation, protein oxidation and depletion of thiols. It is widely accepted that the cytotoxic effect of aflatoxin on normal differentiated cells is due to the production of ROS at high levels [36]. Several reports indicate that aflatoxin administration resulted in excessive lipid peroxidation [4], [37] with concomitant decrease in reduced glutathione (GSH) [7], increased protein

Conclusion

Aflatoxin induced oxidative stress resulting in cytotoxicity in the bone marrow, increased DNA fragmentation, and lipid peroxidation in the liver and testis of rats. In line with this, it induced a significant decrease in GSH in the liver and testis and a significant up-regulation in mRNA expression of the FAS gene in the liver. However, it induced a significant decrease in GSH in liver and testis accompanied with down-regulation in mRNA expression of the PHGPx gene. Both KGE and WPC, alone or

Conflict of interests

The authors declare that there are no conflicts of interests.

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