Vitamin E ameliorates aflatoxin-induced biochemical changes in the testis of mice

R.J. Verma, Anita Nair

Department of Zoology, University School of Sciences, Gujarat University, Ahmedabad-380 009, India

Asian J Androl  2001 Dec; 3: 305-309

Keywords: aflatoxin;  vitamin E; testis; biochemical changes

Abstract

Aim: To assess the effect of aflatoxin on biochemical changes in the testis of mice and the possibility of amelioration by vitamin E treatment. Methods: Adult male albino mice were orally administered with 25 or 50 g of aflatoxin/animal/day (750 or 1500 g/kg body weight) for 45 days. The testis was isolated and processed for biochemical analysis. Results: There was a significant, dose-dependent reduction in DNA, RNA, protein, sialic acid contents and the activities of succinic dehydrogenase, adenosine triphosphatase and alkaline phosphatase in the testis of aflatoxin-treated mice as compared to the vehicle control. However, the acid phosphatase activity was significantly increased in the aflatoxin-treated mice. Vitamin E (2 mg/animal/day) treatment significantly ameliorated the aflatoxin-induced changes, except the acid and alkaline phosphataseactivities in the high dose group. Conclusion: Vitamin E treatment  ameliorates the aflatoxin-induced changes in the testis of mice.

1 Introduction

Aflatoxins are secondary toxic fungal metabolites produced by Aspergillus flavus and A. parasiticus. Aflatoxins are well known hepatotoxic and hepatocarcinogenic agent^[1,2].

Marvan et al^[3] have experimentally studied the distribution of aflatoxin B₁ (AFB₁)  in goslings and chickens. According to the AFB₁ concentration, the organs and tissues were categorized in the order from high to low concentrations as follows: the gonads, the parenchymatous organs (liver and kidney), the lymphopoietic organs (spleen, bursa cloacalis and thymus), the endocrine glands, the muscles, and the lungs, while the brain had the lowest concentration. In Chinese hamsters, Petr and his colleagues^[4]  have shown that after a single intraperitoneal dose of 0.1 mg AFB₁/kg body weight, free AFB₁ has been detected in blood, liver, kidney and testis from minutes up to 8-10 h after injection.

In a previous study^[5], we observed reduced weight of testis, scattered and disorganized cell population in the seminiferous tubules in  mice orally administered with 25 or 50 g of aflatoxin/animal/day for 45 days.  The sperm count and motility were significantly reduced, and a large number of nonviable spermatozoa probably due to loss of membrane integrity was also observed in aflatoxin-treated mice. These alterations were correlated with increased lipid peroxidation in the testis^[6] and were much ameliorated on pretreatment with vitamin E.

2 Materials and methods

2.1 Aflatoxin

Aspergllus parasiticus (NRRL 3240) obtained from the Indian Agricultural Research  Institute, New Delhi, India was grown on sucrose-magnesium sulfate-potasium nitrate-yeast extract (SMKY) liquid medium at 282 for 10 days^[8]. Culture filtrates were extracted with chloroform (1:2, v/v) and passed through anhydrous sodium sulphate. The chloroform extract was evaporated to dryness.

Dried aflatoxin extract was dissolved in fresh chloroform and used for qualitative^[9]  and quantitative^[10]  analysis of aflatoxin. Aflatoxin extract containing B₁, B₂, G₁ and G₂ in the ratio of 8:3:2:1 was used for treating the experimental animals.

2.2 Animals and treatments

Young inbred Swiss strain male albino mice (Mus musculus) weighing 32-34 g were obtained from the Cadila Health Care, Ahmedabad, India. Animals were provided with animal feed (prepared as per the formulation given by the National Institute of Occupational Health, Ahmedabad, India) and tap water ad libitum and maintained under laboratory conditions.

Seventy animals were randomly divided into seven groups of 10 animals each. Group 1 was the untreated control. Animals of Group 2 received olive oil (0.2 mL/animal/day) and Group 3, vitamin E (2 mg/0.2 mL olive oil/animal/day) for 45 days. Animals of Groups 4 and 5 were orally administered with 25 and 50 g aflatoxin/0.2 mL olive oil/animal/day (750 and 1500 g/kg body weight), respectively, for 45 days. Animals of Groups 6 and 7 received aflatoxin (750 and 1500 g/kg, respectively) and vitamin E (2 mg/0.2 mL olive oil/animal/day) 1 h prior to aflatoxin for 45 days.

As already indicated that both aflatoxin and vitamin E were dissolved in the olive oil. As different isomers of aflatoxin exist in the food stuffs, we preferred to carry out the experiment with mixed aflatoxins. The dose of aflatoxin was based on its LD₅₀ value^[11] . The effective dose of vitamin E was based on earlier work^[12]. The duration of treatment (45 days) was based on a possible cumulative toxicity and the duration of spermatogenesis and spermiogenesis^[2,13] in mice.

2.3 Measurements

On completion of the treatment, mice were killed by cervical dislocation. Testis was isolated, blotted free of blood and processed for biochemical analysis. The testicular DNA content was estimated by the method of Giles and Meyer^[14]. The DNA in the supernatant reacts with diphenylamine to give a blue coloured complex whose optical density was measured colorimetrically. The concentration of RNA in the testis was estimated by the method of Mejboum^[15]. The RNA in the supernatant reacts with orcinol reagent to give a greenish colour, which was measured colorimetrically. The concentration of protein was measured by the method of Lowry et al^[16] using bovine serum albumin as the standard. The sialic acid concentration was estimated by the method of Jourdian et al^[17]. The sialic acid was oxidized by periodic acid prior to heating with resorcinol. Free sialic acid gave chromogens which were stable at 0. The chromogen was treated with an organic solvent, which extracted  the colour and was measured spectrophotometrically.

The activity of succinic dehydrogenase (E.C. 1.3.99.1) was measured by the method of Beatty et al^[18] using INT as an electron acceptor. Adenosine triphosphatase (E.C. 3.6.1.3) was measured by the method of Quinn and White^[19]. The inorganic phosphate content released at the end of the reaction was measured by the method of Fiske & Subbarow^[20] . The acid phosphatase (E.C. 3.1.3.2) and alkaline phosphatase (E.C. 3.1.3.1) activities were assayed by the methods of Bessey et al^[21] and Sigma Technical Bulletin No.104^[22].

2.4 Statistics

Data were expressed in meanSEM, if applicable. The Student's t-test was used for statistical analysis and P<0.05 was considered significant.

3 Results  

Oral administration of aflatoxin for 45 days caused a significant, dose-dependent reduction in DNA, RNA, protein and sialic acid contents in the testis of mice compared with the control. Vitamin E treatment significantly ameliorated the aflatoxin-induced changes (Table 1). Table 2 shows the effect of aflatoxin treatment on enzymatic changes in the testis and the amelioration effects of vitamin E treatment. Oral administration of aflatoxin for 45 days caused a significant, dose-dependent reduction in the activities of succinic dehydrogenase, adenosine triphosphatase and alkaline phosphatase, however, the acid phosphatase activity was significantly increased as compared to the vehicle control. Vitamin E treatment caused a significant amelioration in the adenosine triphosphatase, acid phosphatase and alkaline phosphatase activities only in low dose aflatoxin-treated mice. However, a substantial recovery in the succinic dehydrogenase activity was observed in both the low and high dose aflatoxin-treated mice.

Table 1.  Effect of vitamin E treatment on aflatoxin-induced biochemical changes in the testis of mice.
Table 2.  Effect of vitamin E treatment on aflatoxin-induced enzymatic changes in the testis of mice.

4 Discussion

In the present study it was indicated that aflatoxin treatment caused a significant, dose-dependent reduction in the concentration of DNA, RNA and protein in the testis of mice. It is well known that aflatoxin B₁ is activated to AFB₁-8,9-exoepoxide and forms adduct primarily at N₇ position of guanine^[2], thereby reducing its biosynthesis. The decline in protein concentration in the testis of aflatoxin-treated mice could be due to a decline in the protein biosynthesis by forming adducts with DNA, RNA and proteins, an inhibition of RNA synthesis or DNA-dependent RNA polymerase activity, as well as degranulation of endoplasmic reticulum^[1,2].

In addition, oxidative DNA damage and lipid peroxidation are also manifestations of aflatoxin-induced toxicity^[23]. Verma and Nair^[6]   have reported increased lipid peroxidation in the testis of aflatoxin-treated mice. Oxidative stress may also result in the damage of critical cellular macromolecules, including DNA, lipids and proteins^[24]. Renwick et al^[25] reported a concentration-dependent inhibition in protein synthesis in the liver of rat.

During spermatogenesis and spermiogenesis many macromolecules are synthesized^[26].  Sertoli cells secrete both serum proteins and testis-specific proteins, including androgen binding protein, inhibin, Sertoli-derived growth factors and cyclic protein-2^[27]. Therefore, the reduction in these macromolecules could be responsible for the reduction in spermatogenesis and spermiogenesis^[5].

Sialic acid is a sialomucoprotein essential for the maintenance of the structural integrity of the sperm membrane and sperm maturation^[28,29]. Therefore a reduction in the sialic acid concentration in the testis could be responsible for morphological abnormalities observed in spermatozoa.

Succinic dehydrogenase is a key enzyme in the mitochondrial Kreb's cycle, which is mainly concerned with the aerobic oxidation of acetyl CoA and the generation of ATP. A decrease in succinic dehydrogenase activity in the testis of aflatoxin-treated mice indicates a reduction in aerobic oxidation which could be the result of reduced oxygen transport to tissues^[30]. Another possible reason could be due to the accumulation of intracellular calcium, which is known to cause mitochondrial  dysfunction and reduce ATP generation during aflatoxicosis^[31,32]. Roy^[33]  reported mitochondrial swelling during aflatoxicosis. Reduced aerobic oxidation and ATP generation in the testis could be responsible for the reduction in ATPase activity. Reduced succinic dehydrogenase and ATPase activity could explain the reduced sperm count and motility and the increased number of non-viable spermatozoa observed in aflatoxin-treated mice.

Aflatoxin treatment for 45 days caused a significant reduction in the alkaline phosphatase activity in the testis of mice. Alkaline phosphatase is a hydrolyticenzyme, acting on phosphoric esters with the liberation of inorganic phosphate from various substrates. Alkaline phosphatase is mainly involved in passive transport mechanism, suggesting a reduction in the transport mechanism.

Acid phosphatase is very important for tissue reorganization and tissue repair. Intracellularly, acid phosphatase activity is restricted to the lysosomes. Increased acid phosphatase activity in the testis of aflatoxin-treated mice could be due to an increase in the leakage of the enzyme.

Testis is an androgen-dependent organ. The significant reduction in serum testosterone during aflatoxicosis have been reported by Bashandy et al^[34] and Verma and Nair^[35]. 

Therefore, alterations in the testis of aflatoxin-treated mice could be due to a direct effect of aflatoxin on the testis and/or indirectly through reduction in serum testosterone concentration.

Vitamin E treatment significantly ameliorated aflatoxin -induced alterations in the testis of mice. Vitamin E is a potent biological antioxidant. The antioxidative function of vitamin E is mainly due to its reaction with membrane phospholipid bilayers to break the chain reaction initiated by hydroxyl radical^[23].Vitamin E prevents peroxidative changes in the membranes of mitochondria and helps in maintaining the smooth translocation of phosphate ions into mitochondria. Thus oxidative phosphorylation is enhanced. The protective effect of vitamin E on lipid peroxidation in the testis of aflatoxin-treated mice has been reported by Verma and Nair^[6]. In addition, vitamin E has a higher affinity for aflatoxin and acts by reducing its bioavailability through the formation of stable association^[36]. Vitamin E reversal of serum testosterone level in aflatoxin-fed mice could also be a factor for the amelioration in aflatoxin-induced changes^[35].

Acknowledgements

References

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Correspondence to: Dr. R.J. Verma, Department of Zoology, University School of Sciences, Gujarat University, Ahmedabad-380 009, India.
E-mail: zooldeptgu@satyam.net.in
Received 2001-11-06          Accepted 2001-12-05