Introduction

Nuts are rich in an unsaturated fatty acids and various bioactive compounds, which have been including high-quality vegetable protein, minerals, fiber, phytosterols, tocopherols and phenolic compounds. Almonds (Prunusamigdalis), hazelnuts (Corylusavellana), walnuts (Juglansregia), caju (Anacardiumoccidentale), and pistachios (Pistachiavera) are among popularly consumed edible tree nuts.^1,2

Fungi have growing on nuts which were formed the mycotoxin and can be decrease the quality of nuts by reducing their nutritive quality.³ Both of the mycotoxins and aflatoxin have been formed by the molds Aspergillus flavus and Aspergillus parasiticus. Blue 1 (B1), B2, Green 1 (G1), and G2 are the most common aflatoxin strains that were found in foods, thus named because of their fluorescence properties and chromatography patterns.⁴ Optimal aflatoxin production occurs at temperatures nearly to 30 °C.⁵

Mammals convert aflatoxins into the  M1 and M2 metabolites, which are carcinogenic.^6,7 Aflatoxin contamination usually occurs either by slowly acquiring aflatoxin over time in smaller quantities or by consuming large amounts at once. Aflatoxin contamination can lead to a variety of health problems, including cancers, mental and digestive problems, hemorrhages, and malabsorption.⁸

Aflatoxins reduce growth and suppress immune functions in animals. Aflatoxin induces hepatic and renal tumors in rodents, and has been implicated in esophageal cancer. Humans are exposed to mycotoxins through the consumption of contaminated foods, as well as by exposure to dust and air containing these toxins. Aflatoxins are hepatocarcinogenic, predominantly in conjunction with chronic hepatitis B virus infection, and form aflatoxicosis in episodic poisoning outbreaks.^9,10

Here we have been evaluated the potential for yogurt and selenium to ameliorate aflatoxin-contaminated nut-induced hepatotoxicity in rats.

Materials and Methods

Nuts

Five kilograms of commonly consumed nuts (pistachio, caju, walnut, almond, and hazelnut) were obtained from a local market of Riyadh in the Kingdom of Saudi Arabia. These nuts are more susceptible to mold growth.

Reagents

All the materials used for this experiment were of analytical grade and used without further purifications. BioMeriuex Kits were purchased from Alkan Co. for Chemicals and Biodiagnostics. Selenium was obtained from Sigma-Aldrich.

Probiotic Bacteria

Lactobacillus delbrueckiisubspeciesbulgaricusCH-2 and Streptococcus thermophilus ST-36 were obtained from the HansenLab (University of Denmark).

Experimental animals

Fifty adult male Sprague-Dawley strain (albino) rats, weighs within 130–140g, were provided by the experimental animals of the center in Research Center in Prince Sultan Military Medical City, Riyadh. Rats were housed as groups in wire cages under laboratory conditions and, during a 1-week adaption period, were fed a standard diet. Food and water were provided adlibitum. Animal management ethical guidelines were followed throughout the study and permission was obtained from the concerned department.

Standard diet

The standard experimental diet was composed of corn starch(598 g kg^-1), casein (200 g kg^-1), soybean oil (100 g kg^-1), vitamin mixture (10 g kg^-1), salts mixture (40 g kg^-1), cellulose (50 g kg^-1), and choline chloride (2 g kg^-1), according to Ref.¹¹

Preparation of ordinary yogurt

L.delbrueckii subspecies bulgaricus CH-2 was cultured in MRS broth at 37 °C for 24 h. S. thermophilus ST-36 was grown in M17 broth at 40 °C for 24 h. Whole milk was heated to boiling temperature to reduce its volume by approximately 20% after cooling;this milk was then heated to 90 °C for 5 min and then cooled to 42°C and inoculated with 1% of L.delbrueckii sub-species bulgaricus CH-2 and S. thermophilusST-36, then incubated at 40°C until coagulation (about 4 h).¹²

Nuts storage and quantification of aflatoxin content

Raw nuts were stored in glass dishes at 25 ^oC and 60% relative humidity for 6 months. Then, these nuts were crushed to estimate their aflatoxins content, as previously described.^13,14 The total aflatoxins were 23.25, 23.66, 22.07, 26.02 and 28.6μgkg^-1 in the pistachios, caju, walnuts, almonds, and hazelnuts, respectively. Mixed crushed nuts were added as 3%(w/w) to the standard diet, with consideration of the nutritional value of the nuts.

Treatment schedule

Rats were divided into five groups, 10 rats per group, as follows: (1)  normal control group fed the standard diet; (2) positive control group fed the standard diet plus aflatoxin-contaminated nuts to induce hepatotoxicity, as reported in previous work;^9,10 (3) selenium group fed the standard diet plus aflatoxin-contaminated nuts plus selenium by stomach tube (3 mg kg^-1 body weight); (4) yogurt group, fed the standard diet plus aflatoxin-contaminated nuts plus yogurt by stomach tube (160 mlkg^-1 body weight); (5) yogurt plus selenium group, fed the standard diet plus aflatoxin-contaminated nuts plus selenium and yogurt by stomach tube (3 mg kg^-1 and 160 mlkg^-1 body weight, respectively).

The daily food intake and weekly body weight of the rats were recorded. The feed efficiency ratio (FER) was calculated using the method of Ref.¹⁵ After completion of the experimental period (60 days), rats were fasted overnight and sacrificed to obtain blood and liver samples for biochemical analyses.

Serum analyses

From these samples, we quantified the serum alanine and aspartate aminotransferase (ALT +AST), alkaline phosphatase (ALP), and gamma-glutamyltransferase (γGT) enzyme activities. Also, serum total protein,  globulin, albumin, creatinine, uric acid, urea, and bilirubin contents were enzymatically determined as mentioned in the method of Ref.¹⁶ The activity of glutathione peroxidase (GPX), malondialdehyde (MDA), and superoxide dismutase(SOD) enzymes were determined as previously described.^17–19

Liver biochemical estimations

Livers of rats were rapidly removed and parts of them perfused with 50 to 100 ml of ice-cold 0.9% NaCl solution for estimation of the liver glutathione peroxidase (GPX), superoxide dismutase (SOD) and malondialdehyde (MDA) activities according to Refs.^18,20,21

Statistical analysis

Data were subjected to ANOVA. Comparison of means was performed using Duncan’s multiple-range test with level of significance 0.05, complemented by Kruskal-Wallis correlation method to analyze correlations between parameters at significance levels of 0.05.

Results and Discussion

Herein, we found that rats fed aflatoxin-contaminated nuts have been decreased the body weight gain and FER, but that selenium or yogurt, or both, could ameliorate these effects. Food intake did not vary among the five test groups (Table 1). It has been well-documented nut contamination with aflatoxins (from A. flavusand A. parasiticus) is a major health concern, especially in hot and humid regions. Under conventional storage conditions, aflatoxin-producing molds are able to grow exponentially.²³Aflatoxins have a low molecular weight therefore quickly absorbed in the gastrointestinal tract and appear as metabolites in theblood.^24,25 Following exposure to aflatoxins, they can be detected covalently bound to DNA, thereby reducing protein synthesis; this effect can be persist for up to 5 days. Also, animals exposed to aflatoxin are less efficient at food use and dietary animals exposed to aflatoxin have a reduced growth rate and productivity. These effects are likely due to an increased degradation of lipids and proteins.^26,27

Co-administration of selenium and yogurt has made improvement in the nutritional results. Indeed, Navarro-Alarcon and Cabrera-Vique have been detected the increasing in growth and development of the selenium supplementation.²⁸ Fermented milk and yogurt are considered safe and nutritious. Yogurt is related to its component and proteolytic bacteria. Milk is a source of protein, calcium, and the B-group vitamins, as well as vitamin A, vitamin C, magnesium, and zinc.²⁹ The consumption of fermented dairy products containing lactic acid bacteria, as well as the probiotic bacteria which was found in yogurt. These can be proposed to reduce the risk of liver cancer by binding to the mutagens produced by intestinal bacteria.^30,31

Table 1: Mean values ± standard deviation (SD) of body weight gain, food intake, and feed efficiency ratio (FER)of the control group, rats fed aflatoxin-contaminated nuts, and those supplemented with selenium, yogurt or both selenium and yogurt.

Values with the different letters indicate significant difference (P<0.05) and vice versa.

Because of the liver’s role in the detoxification of environmental xenobiotics, consuming aflatoxins-contaminated nuts can cause liver injury and induce hepatotoxicity, which is agreement with our results. The group fed aflatoxins-contaminated nuts had increased serum ALT, AST, ALP, and γGT enzyme activities, compared to the control group. Supplemental selenium or yogurt, or both, could significantly lower these enzyme activities to control levels (Table 2). Multiple studies reported that aflatoxins, potent hepatotoxics, and hepatocarcinogenic mycotoxincan induce lipid peroxidation. These compounds have been also associated with various diseases, including aflatoxicosis and hepatocellular carcinoma.^27,32 Here, we demonstrate that the consumption of aflatoxin-contaminated nuts by rats forced a marked elevation in liver enzyme activities, demonstrating hepatocellular damage, as previously reported.³³ Selenium tended to alleviate serum liver enzymes.^34,35 The improvement in the liver enzyme levels followed by the consumption of yogurt, that is likely related to the yogurt’s probiotic organisms. Therefore, directly affect multiple processes, including digestion and immune function.^36,37

Table 2: Mean values ± standard deviation (SD)of serum ALT, AST, ALP and γ GT enzymes of the control group, rats fed aflatoxin-contaminated nuts, and those supplemented with selenium, yogurt or both selenium and yogurt.

Values with the different letters indicate significant difference (P<0.05) and vice versa.

Compared to the control group, the consumption of aflatoxin-contaminated nuts has been reduced the total protein and elevated the creatinine, urea, uric acid, and bilirubin levels. Supplemental selenium decreased globulin and increased creatinine, urea, and bilirubin levels, whereas yogurt increased urea compared to the control group. Compared to the group fed aflatoxin-contaminated nuts, selenium or yogurt, or both, significantly increased serum total protein and decreased creatinine, urea, uric acid, and bilirubin levels (Table 3). Uric acid is the metabolic end product of purine metabolism. The observed decreasing level of uric acid in the treated groups is likely a result of an increased utilization of uric acid, thereby inhibiting the generation of free radicals.²⁷ Selenium can reduce nephrotoxicityby attenuating oxidative-stress-associated kidney injury through the reduction of oxygen free radicals and lipid peroxidation in rats treated with gentamicin. Selenium is a cofactor of several enzymes that participate in the regulation of enzymatic antioxidant defenses.^38,39 Here we show that improvement of this renal parameter is related to the bioactive peptides that are generated during the production of fermented dairy products, which have antioxidative and growth promoting properties.^40–42

Table 3: Mean values ± standard deviation (SD) of serum total protein, albumin, globulin, creatinine, urea, uric acid and bilirubin of the control group, rats fed aflatoxin-contaminated nuts, and those supplemented with selenium, yogurt or both selenium and yogurt.

Values with the different  letters indicate significant difference (P<0.05) and vice versa

Also, the aflatoxin-contaminated nut-fed group had decreased serum and liver GPX and SOD enzyme activities and increased serum and liver MDA, as compared to the control group. Selenium or yogurt, or both, was able to normalize serum and liver GPX, SOD, and MDA levels (Table 4). In the current study, consumption of aflatoxin-contaminated nuts induced oxidative damage through the generation of reactive oxygen species. This was accompanied by an increase in the expression of liver enzymes and decreased biological activities of some liver antioxidant enzymes. Glutathione is exhausted by glutathione-related enzymes to detoxify the peroxides formed from increased lipid peroxidation under oxidative stress. The increase in hepatic MDA level might be because aflatoxins are metabolized by the cellular cytochrome P450 enzyme system to form the reactive intermediate, aflatoxin-8,9-epoxide, which in-turn reacts with macromolecules, such as lipids and DNA. This leads to cellular injury and lipid peroxidation.⁴³ Also, MDA was elevated in the treatment groups compared to the control group, most possibly reflecting an adaptive reaction towards free radical damage in the liver.⁴⁴

Table 4: Mean values ± SD of serum and liver GPX, SOD and MDA of the control group, rats fedaflatoxin-contaminated nuts, and those supplemented with selenium, yogurt or both selenium and yogurt.

Values with the different letters indicate significant difference (P<0.05) and vice versa

Selenium’s ability to enhance GPX activity might be because of increased selenium bioavailability, thereby preventing the formation of free radicals and protecting both integrity and functions of tissues, thus protecting the liver from peroxidation of  lipid and changes in glutathione and antioxidant enzyme activities.^45,46 The administration of the lactic acid bacteria in yogurt results in increased antioxidative enzyme activity and modulated circulatory oxidative stress, thereby protecting the cells against damage.^31,42,47

Conclusion

Selenium and yogurt reduce the hepatotoxicity in rats caused by consuming gaflatoxin-contaminated nuts. Further studies are now needed to better understand the in vivo mechanism(s) by which selenium and yogurt reduce aflatoxin toxicity.

Acknowledgements

I would like to record my profound gratitude to Princess Nourah bint Abdulrahman University for its moral support in accomplishing this research paper.

Competing interest

I declare that there is no conflict of interest regarding the publication of this paper which titled “Lowering effect of selenium and yoghurt on nuts contaminated with aflatoxins induced hepatotoxicity in rats.”

Funding sources

I confirm that The funding of this research was on my personal account .

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