Home  |  Archive  |  AJA @ Nature  |  Online Submission  |  News & Events  |  Subscribe  |  APFA  |  Society  |  Links  |  Contact Us  |  中文版

Ameliorative effect of vitamin E on aflatoxin\|induced lipid peroxidation in the testis of mice

R.J. Verma,  Anita Nair

Department of Zoology, University School of Sciences, Gujarat University, Ahmedabad 380009, India

Asian J Androl  2001 Sep; 3: 217-221

Keywords: aflatoxins; vitamin E; testis; lipid peroxidation
Abstract

Aim: To evaluate the ameliorative effect of vitamin E on aflatoxin-induced lipid peroxidation in the testis. Methods: Adult male albino mice were orally administered 25 or 50  g of aflatoxin in 0.2 mL olive oil per d for 45 d. The testis was isolated, blotted free of blood and processed for biochemical analysis. Results: There was a dose-dependent significantly higher lipid peroxidation in the testis of aflatoxin treated mice than in the controls. The levels of non-enzymatic antioxidants such as glutathione, total and reduced ascorbic acid, as well as the activities of enzymatic antioxidants, such as superoxide dismutase, glutathione peroxidase and catalase were significantly lower in the testis of aflatoxin treated mice. Vitamin E (2 mg/d per animal; orally) pretreatment significantly ameliorates the aflatoxin-induced lipid peroxidation which could be due to higher enzymatic and non-enzymatic antioxidants in the testis of mice as compared with those given aflatoxin alone. Conclusion: Vitamin E pretreatment significantly ameliorates aflatoxin-induced lipid peroxidation in the testis of mice.

1 Introduction

Aflatoxins are secondary toxic fungal metabolites produced by Aspergillus flavus and A. parasiticus. They not only contaminate our food stuffs but are also found in edible tissues, milk and eggs after consumption of contaminated feed by farm animals. In utero exposure of aflatoxin through mother's blood has also been reported in human beings[1-2].

Aflatoxins are well known for its hepatotoxic and hepatocarcinogenic effects[3]. Aflatoxin B1 (AFB1) is activated to AFB1-8,9-oxide and forms adduct primarily at N7 position of guanine and is responsible for its mutagenic and carcinogenic effects[4,5]. In addition, lipid peroxidation and oxidative DNA damage are also the manifestations of aflatoxin B1-induced toxicity. Souza et al[6] have reported significant rise in lipid peroxidation in the liver of rats 72 hours after a single intraperitoneal dose of AFB1. Histological changes such as hepatocellular necrosis and bile duct proliferation were also observed. All these changes were markedly inhibited in animals pretreated with vitamin E. Shen et al[7] have also reported that vitamin E treatment significantly inhibited aflatoxin B1-induced lipid peroxidation in rat liver.

In the previous study we reported derangement of seminiferous tubules in the testis of aflatoxin-treated mice[8]. A significant reduction in epididymal sperm motility, count and fertility rate was also observed. This may be due to oxidative stress which is generally correlated with cellular damage[6,9]. However, the effect of aflatoxin on lipid peroxidation in the testis remains unknown.

The aim of the present investigation was to evaluate lipid peroxidation and antioxidative defence mechanisms in the testis of aflatoxin-treated mice. The possible role of vitamin E in the amelioration of aflatoxin-induced lipid peroxidation was also investigated.
2 Materials and methods

2.1  Aflatoxin

Aspergillus parasiticus (NRRL 3240) obtained from the Indian Agricultural Research Institute, New Delhi, India, was grown on sucrose-magnesium sulfatepotassium nitrate-yeast extract (SMKY) liquid medium at (282) for 10 d [10]. Culture filtrates were extracted with analytical grade chloroform (1:2, v/v) and passed through a bed of anhydrous sodium sulphate. The chloroform extract was evaporated to dryness and stored.

Both the residue and the dried aflatoxin extract were dissolved separately in fresh chloroform for chemical analysis. Crude aflatoxin (100 L) extracted from Aspergillus parasiticus culture medium was first fractionated on silica gel G coated activated TLC plates along with aflatoxin standard (a gift from the International Agency for Research on Cancer, Lyon, France). The plates were developed in a solvent consisting of toluene: isoamyl alcohol: methanol (90:32:2, v/v)[11]. The air-dried plates were observed under long-wave UV light (360 nm) for aflatoxins. Different components of aflatoxin were initially identified visually by comparing the colour and intensity of fluorescence as well as polarity of sample spots with standards. Aflatoxin B1 and B2 showed blue fluorescent spots; aflatoxin G1 and G2 showed bluish green fluorescent spots. The order of appearance from lesser to greater Rf was: aflatoxin G2, G1, B2 and B1.

Chemical confirmation of aflatoxin was done by spraying trifluoroacetic acid (TFA) and 25 % sulfuric acid[12]. The aflatoxin positive extracts (confirmed through visual observation) were spotted on another TLC plate. A very little amount of TFA was directly applied on to sample spots. The standard aflatoxin was also treated similarly and plates were developed in the solvent system as described earlier. Aflatoxin B1 and G1 fluoresced at reduced Rf value than the untreated spot. Spray plate with 25 % sulfuric acid was observed under long-wave UV light (360 nm). Aflatoxins showed yellow fluorescent spots.

Each spot (from freshly run TLC plates) was scraped separately, dissolved in chilled methanol and subjected to spectrophotometric analysis at 360 nm[13]. The concentrations of aflatoxins were calculated using molar extinction coefficients. Other isomers were non-detectable.

Dried aflatoxin extract containing B1, B2, G1 and G2 in the ratio of 8:3:2:1, respectively, was used for treating the experimental animals in olive oil carrier. Under natural condition different isomers of aflatoxins exist together in the form of mixture, we preferred to carry out the experiments with mixed aflatoxins only which is more pertinent from the point of view of application.

2.2 Animals and treatments

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

Seventy male mice were randomly divided into seven groups and caged separately. Group 1 (untreated control) animals were maintained without any treatment. Animals of Group 2 and 3 received olive oil (0.2 mL/d per animal) and vitamin E (2 mg/0.2 mL olive oil/d per animal), respectively, for 45 d and served as positive controls. Animals of Group 4 and 5 were orally administered with aflatoxin chloroform extract (25 and 50 g/0.2 mL olive oil/d per animal, or 750 and 1500 g/kg body weight, respectively) for 45 d. Group 6 and 7 animals were orally administered with vitamin E (2 mg vitamin E/0.2 mL olive oil/d per animal) 1 hour prior to the administration of aflatoxin as indicated in Group 4 and 5 for 45 d. Olive oil was obtained from Figaro, Madrid, Spain and Vitamin E from HiMedia Pvt. Ltd., Mumbai, India. Aflatoxin and vitamin E were dissolved in olive oil. Hence it was used as a vehicle in Group 2. The dose of aflatoxin was determined on the basis of its LD50 value, i.e., 9 mg/kg body weight[14]. The effective dose of Vitamin E was based on earlier work[15]. All the treatments were given orally using a feeding tube attached to a hypodermic syringe. Duration of the treatment (45 d) was based on cumulative toxicity of aflatoxin[16] and spermatogenic and spermiogenic cycle in mice[17].

2.3 Measurements

On completion of the treatment, mice were killed by cervical dislocation. The testis was isolated, blotted free of blood and processed for biochemical analysis. The levels of lipid peroxidation was measured by quantification of thiobarbituric acid (TBA)-reactive substance in accordance with the method described by Ohkawa et al[18]. The activities of superoxide dismutase (SOD) (E.C.1.15.1.1.) and glutathione peroxidase (E.C.1.11.1.9) were measured according to the method of Kakkar et al[19] and Pagila and Valentine[20], respectively. Catalase (E.C. 1.11.1.16) activity was measured by the method of Luck[21] using hydrogen peroxide (H2O2) as a substrate. All enzyme activities were measured at 37. The levels of glutathione (GSH) was assayed by the method of Grunert and Philips[22]. The levels of ascorbic acid (total, dehydro- and reduced) was determined by the method of Roe and Kuether[23]. All the samples were analysed for protein content by the folin phenol method of Lowry et al[24] using bovine serum albumin as standard.

2.4 Statistics

Student's t-test was used for statistical analysis of the data and P<0.05 was considered significant.

3 Results  

3.1 In situ TUNEL staining

Table 1 shows that aflatoxin treatment for 45 d caused a dose-dependent higher level of lipid peroxidation in the testis of mice than in the controls (P<0.01). The levels of non-enzymatic antioxidants such as glutathione, total and reduced ascorbic acid, as well as, the activities of enzymatic antioxidants (superoxide dismutase, glutathione peroxidase and catalase) were significantly lower in the testis of aflatoxin treated mice compared to the controls (P<0.01). However, significantly higher level of dehydroascorbic acid was observed in the testis of the aflatoxin treated mice (P<0.01).

Vitamin E pretreatment significantly prevents aflatoxin-induced lipid peroxidation which could be due to higher enzymatic and non-enzymatic antioxidant levels in the testis of mice compared with those given aflatoxin alone.  

Table 1. Effect of pretreatment with vitamin E on aflatoxin-induced lipid peoxidation and antioxidative defence mechanisms in the testis of mice (s,  n=10).

Parameters

Experimental Groups

1

2

3

4

5

6

7

Lipid peroxidation (n moles 
malondialdehyde/mg protein)

74.630.73

74.750.89

74.400.71

96.731.45c

133.911.30c

85.571.91f

120.240.83i

Superoxide dismutase activity(Units/mg protein)

0.190.01

0.200.07

0.190.002

0.140.002c

0.070.008c

0.170.002f

0.120.005i

Glutathione peroxidase activity (n moles of NADPH consumed/mg  protein/min)

10.280.20

10.060.11

10.530.13

6.390.11c

3.480.17c

8.990.14f

5.110.009i

Catalase activity (moles of H2O2 consumed/mg protein/min)

20.150.30

21.370.28

20.270.40

12.410.52c

8.990.19c

18.650.46f

11.490.23i

Glutathione (g GSH/g tissue weight)

50.840.42

51.000.62

50.720.21

32.990.50c

20.130.75c

44.610.39f

27.990.44i

Total ascorbic acid (mg/g tissue weight)

4.930.10

4.800.15

4.520.19

2.350.10c

1.870.009c

4.770.006f

2.460.004i

Dehydroascorbic acid (mg/g tissue weight)

1.600.12

1.690.009

1.580.13

2.190.008c

2.880.12c

1.870.007f

2.610.004i

Reduced ascorbic acid (mg/g tissue weight)

3.350.004

3.280.007

3.400.003

1.560.001c

1.190.005c

2.980.002f

2.400.003i

Group 1: Untreated control;   
Group 2: Olive oil control;  
 Group 3: Vitamin E control;  
 Group 4: AF-25   g treated;   
 Group 5: AF-50   g treated; 
 Group 6: AF-25   g treated+VitE; 
 Group 7: AF-50   g treated+VitE;
cP<0.01, compared to the control; fP<0.01,  compared to Group 4; iP<0.01, compared to Group 5.

4 Discussion

The higher lipid peroxidation observed in the present investigation could be due to a lower antioxidant capacity of the cells (Table 1). Oxidative stress occurs in a cell or tissue when the concentration of reactive oxygen species (ROS) generated exceeds the antioxidant capability of that cell[25]. Aflatoxins can produce ROS by either direct or indirect mechanisms[26]. Oxidative stress can also occur when there is a decrease in the antioxidant capacity of a cell[27]. It is also known that aflatoxins induce the formation of enzymes involved in ROS metabolism[28]. The levels of enzymatic antioxidants (SOD, glutathione peroxidase and catalase) and non-enzymatic antioxidants (vitamin C, glutathione, vitamin E) are the main determinants of the antioxidant defence mechanism of the cell. While SOD has been recognized to play an important role in the defence mechanism of the body against harmful effects of oxygen free radical in the biological systems, two related enzymes, glutathione peroxidase and catalase scavenge the dismutation of the superoxide radicals.

During radical scavenging action, ascorbic acid is suggested to be transformed into dehydroascorbate. Reduced glutathione is required for the conversion of dehydroascorbate back to ascorbate. The fall in the level of reduced glutathione influences this back-conversion and may explains the lower level of ascorbic acid in the aflatoxin treated animals in the present study.

Verma et al[29] have shown a slightly higher intracellular calcium in the testis of rabbits. Castilho et al[30] proposed that calcium and prooxidant significantly reduced mitochondrial glutathione and NADPH, substrate of the antioxidant enzymes glutathione peroxidase and glutathione reductase, respectively, which favours the accumulation of H2O2. Hoehler et al[31] observed that the lack of an adequate supply of NADPH and GSH to permit H2O2 consumption by the GSH-dependent glutathione peroxidase and NADPH-dependent glutathione reductase, together with an increased concentration of free iron within the cell stimulate the production of hydroxyl radical via a Fenton reaction due to mobilization of ferrous by calcium.

The decline in these enzyme activities could be due to a decline in protein biosynthesis by forming adducts with DNA, RNA and protein and inhibits RNA synthesis and DNA-dependent RNA polymerase activity as well as causing degranulation of endoplasmic reticulum[32,33]. In addition, oxidative stress may result in damage to critical cellular macromolecules including DNA, lipids and proteins[34]. Both oxygen radicals and peroxides are able to inactivate antioxidant enzymes[35]. Baumber et al[36] reported that hydrogen peroxide is the major ROS responsible for damage to equine spermatozoa. The decrease in sperm motility associated with ROS occurs in the absence of any detectable decrease in viability, acrosomal integrity or mitochondrial membrane potential or of any detectable increase in lipid peroxidation.

Vitamin E pretreatment significantly lowered the aflatoxin-induced lipid peroxidation in the testis. The protective effects of vitamin E on lipid peroxidation has also been reported by Shen et al[7] in rat liver and by Cassand et al[37] in in vitro studies. The protective effect of vitamin E against lipid peroxidation could be due to a significant recovery in the antioxidant capacity of the cell (Table 1). 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[7]. Ibeh and Saxena[38] reported significant alterations in testicular sorbitol dehydrogenase, lactic dehydrogenase, glucose-6 phosphate dehydrogenase, gamma glutamyl transpeptidase, reduced the quality of sperm and the marked pathological changes in the testis of rats given aflatoxin B1 alone. Alpha-tocopherol (vitamin E) supplementation increased the blood level of aflatoxin. A reduction in toxicity of free radical by alpha-tocopherol in association with a reduction in aflatoxin metabolism seems to be responsible for the protective influences.

Also, vitamin E has a high affinity for aflatoxin and acts by reducing the bioavailability of aflatoxin after forming stable association with it[39].

The above report elucidates that vitamin E pretreatment significantly inhibited aflatoxin-induced lipid peroxidation due to increased antioxidant capacity of the cell.

Acknowledgments

Financial assistance received from the University Grants Commission, New Delhi, India is thankfully acknowledged. The authors are grateful to Dr M.D. Friesen of the International Agency for Research on Cancer, Lyon, France, for providing pure aflatoxin samples.

References

[1] Pohland AE. Mycotoxin in review. Food Addt Contam 1993; 10: 17-28.
[2] Fink GJ. Mycotoxins: their implications for human and animal health. Vet Q 1999; 21: 115-20.
[3] Wogan GN. Aflatoxin as a human carcinogen. Hepatology 1999; 30: 573-5.
[4] Wang JS, Groopman JD. DNA damage by mycotoxins. Mutat Res 1999; 424: 167-81.
[5] Denissenko MF, Cahill J, Kondriakova TB, Gerber N, Pfeifer GP. Quantitation and mapping of aflatoxin B1-induced DNA damage in genomic DNA using aflatoxin B1-8,9-epoxide and microsomal activation systems. Mutat Res 1999; 425: 205-11.
[6] Souza MF, Tome AR, Rao VS. Inhibition by the bioflavonoid ternatin on aflatoxin B1-induced lipid peroxidation in rat liver. J Pharm Pharmacol 1999; 51: 125-9.
[7] Shen HM, Ong CH, Lee BL, Shi CY. Aflatoxin B1-induced 8-hydroxydeoxyguanosine formation in rat hepatic DNA. Carcinogenesis 1995; 16: 419-22.
[8] Nair A, Verma RJ. Effect of aflatoxin on testis of mouse and amelioration by vitamin E. Indian J Toxicol 2000;  7: 109-16.
[9] Romero FJ, Bosch-Morell F, Romero MJ, Jareno EJ, Romero B, Marin N, et al. Lipid peroxidation products and antioxidants in human disease. Environ Health Perspect 1998; 106 Suppl 5: 1229-34.
[10] Diener UL, Davis ND. Aflatoxin production by isolates of Aspergillus flavus. Phytopathology 1966; 56: 1390-3.
[11] Reddy TV, Vishwanathan L, Venkitasubramanian TA. Thin layer chromatography of aflatoxins. Anal Biochem 1970; 38: 568-71.
[12] Stack ME, Pohland AE. Collaborative study of a method for chemical confirmation of the identity of aflatoxins. J Assoc Off Anal Chem 1975; 58: 110-3.
[13] Nabney J, Nesbitt BF. A spectrophotometric method for determining the aflatoxins. Analyst 1965; 90: 155-60.
[14] Smith JE, Moss MO. Mycotoxins\|formation, analysis and significance. New York: John Wiley and Sons Ltd; 1985.
[15] Chinoy NJ, Sharma AK. Amelioration of fluoride toxicity by vitamin E and D in reproductive functions of mice. Fluoride 1998; 31: 203-18.
[16] Groopman JD, Cain LG, Kensler TW. Aflatoxin exposure in human populations: Measurements and relationship to cancer. CRC Critical Rev Toxicol 1988; 19: 113-45.
[17] Lamming GE. Marshall's physiology of reproduction. v2, Reproduction in male, 4th ed.  Edinburgh:Churchill Livingstone; 1990.
[18] Ohkawa H, Ohishi N, Yagi K. Assay for lipid peroxides in animal tissues by thiobarbituric acid reaction. Anal Biochem 1979; 95: 351-8.
[19] Kakkar P, Das B, Viswanathan PN. A modified spectrophotometric assay of superoxide dismutase. Indian J Biochem Biophys 1984; 21: 130-2.
[20] Pagila DE, Valentine WN. Studies on the quantitative and qualitative characterization of erythrocyte glutathione peroxidase. J Lab Clin Med 1967; 70: 158-69.
[21] Luck H. A spectrophotometric method for the estimation of catalase, in Methods of enzymatic analysis. New York: Academic Press; 1963. p 886-7.
[22] Grunert RR, Philips PH. A modification of the nitroprusside method for analysis of glutathione. Arch Biochem 1951; 30: 217-25.
[23] Roe JH, Kuether CA. The determination of ascorbic acid in whole blood and urine through the 2,4-dinitrophenylhydrazine derivatives of dehydro-ascorbic acid. J Biol Chem 1943; 147: 399-407.
[24] Lowry OH, Rosebrough NJ, Farr AL, Randall RJ. Protein measurement with the folin phenol reagent. J Biol Chem 1951; 193: 265-75.
[25] Sies H. Oxidative stress: introduction. In: Oxidative Stress: Oxidants and Antioxidants.San Diego, California: Academic Press; 1991. p 15-22.
[26] Halliwell B. Mechanisms involved in the generation of free radicals. Pathol Biol 1996; 44: 6-13.
[27] Trush MA, Kenslar TW. An overview of the relationship between oxidative stress and chemical carcinogenesis. Free Radic Biol Medicine 1991; 10: 201-9.
[28] Singh N, Clausen J. Different tissue responses of mixed function oxidases and detoxifying enzymes to aflatoxin B1 administration in the rat. Br J Exp Path 1980; 61: 611-6.
[29] Verma RJ, Kolhe AS, Chaudhari SB. Intracellular calcium accumulation during aflatoxicosis. Med Sci Res 1998; 26: 339-41.
[30] Castilho RF, Kowaltowski AJ, Menicke AR, Bechara EJH, Vercesi AE. Permeabilization of the inner mitochondrial membrane by Ca2+ ions is stimulated by L-butyl hydroperoxide and mediated by reactive oxygen species generated by mitochondria. Free Rad Biol Med 1995; 18: 479-86.
[31] Hoehler D, Marquardt RR, McIntosh AR, Xiao H. Free radical generation as induced by ochratoxin-A and its analogs in bacteria (Bacillus brevis). J Biol Chem 1996; 271: 27388-94.
[32] Cullen JM, Newberne PM. Acute hepatotoxicity of aflatoxins inToxicol. Aflatoxins. In: Eaton  DL, Groopman JD, editors. San Diego, California: Academic Press; 1994. p 3-26.
[33] Groopman JD, Wang JS, Scholl P. Molecular biomarkers for aflatoxins: From adducts to gene mutations to human liver cancer. Can J Physiol Pharmacol 1996; 74: 203-9.
[34] Breimer LH. Molecular mechanisms of oxygen radical carcinogenesis and mutagenesis: the role of DNA base damage. Mol Carcinogenesis 1990; 3: 188-97.
[35] Pigeolet E, Corbisier P, Houbion A, Lambert D, Michiels C, Raes M, et al. Glutathione peroxidase, superoxide dismutase and catalase inactivation by peroxides and oxygen derived free radicals. Mech Ageing Dev 1990; 51: 283-97.
[36] Baumber J, Ball BA, Gravance CG, Medina V, Davis-Morel MC. The effect of reactive oxygen species on equine sperm motility, viability, acrosomal integrity, mitochondrial membrane potential and membrane lipid peroxidation. J Androl 2000: 21: 895-902.
[37] Cassand P, DeCoudu S, Leveque F, Daubeze M, Narbonne JF. Effect of vitamin E dietary intake on in\|vitro activation of aflatoxin B1. Mutation Res 1993; 319: 309-16.
[38] Ibeh IN, Saxena DK. Effect of alpha-tocopherol supplementation on the impact of aflatoxin B1 on the testis of rats. Exp Toxicol Pathol 1998: 50: 221-4.
[39] Odin AP. Vitamins as antimutagens: Advantages and some possible mechanisms of antimutagenic action. Mutation Res 1997; 386: 39-67.

home

Correspondence to:  Dr. Ramtej J. Verma, Department of Zoology, University School of Sciences, Gujarat University, Ahmedabad 380009, India.
Tel: +91-079-630 2362 (O)
E-mail: zooldeptgu@satyam.net.in
Received 2000-12-01                Accepted 2001-07-09