Protective effect of vitamin E against mercuric chloride reproductive toxicity in male mice

Reproductive Toxicology 15 (2001) 705–712

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Protective effect of vitamin E against mercuric chloride reproductive toxicity in male mice Mandava V. Rao*, P.S.N. Sharma Reproductive Endocrinology and Toxicology Division, Department of Zoology, School of Sciences, Gujarat University, Ahmedabad 380009, India Received 26 April 2001; received in revised form 26 July 2001; accepted 4 August 2001

Abstract Mercury intoxication has been associated with male reproductive toxicity in experimental animals and mercury may have the potential to produce adverse effects on fertility in men. Vitamin E may protect against toxic effects of mercury in the liver and other tissues. To investigate the protective role of vitamin E against mercuric chloride toxicity for the testis, epididymis, and vas deferens of adult male mice, animals were treated with either mercuric chloride 1.25 mg/kg/day, vitamin E 2 mg/kg/kg, or a combination of the two treatments. Control animals were treated with water. Treatments were administered by daily gavage for 45 days. An additional group of animals treated with mercuric chloride were permitted to recover for 45 days after mercuric chloride treatments. Parameters studied included serum testosterone, epididymal sperm count, motility, and morphology, epididymal and vas deferens adenosine triphosphatase (ATPase), phosphorylase, sialic acid, glycogen and protein, testicular succinate dehydrogenase (SDH), phosphatases, cholesterol, ascorbic acid, and glutathione. Fertility was evaluated by sperm positive vaginal smears after overnight cohabitation with a female. Mercuric chloride produced a reduction in epididymal sperm count, sperm motility, and sperm viability, and there were no sperm-positive smears in this group. Biochemical tests from the male reproductive organs were also altered by mercuric chloride treatment. Coadministration of vitamin E with mercuric chloride prevented the changes in sperm and biochemical parameters and was associated with control rates of sperm positive smears after cohabitation. Animals given vitamin E with mercuric chloride also had lower concentrations of mercury in the testis, epididimyis, and vas deferens. Permitting animals to recover for 45 days after mercuric chloride treatment resulted in partial recovery of sperm and biochemical parameters. Vitamin E cotreatment has a protective role against mercury-induced male reproductive toxicity. © 2001 Elsevier Science Inc. All rights reserved. Keywords: Mercuric chloride; Vitamin E; Sperm; Fertility; Reproductive organs; Mice

1. Introduction The industrial use of mercury and its general toxic effects on human and animal systems are well known [1,2]. Mercury compounds are known to affect testicular spermatogenic and steroidogenic functions in experimental animals and men [3]. Mercury intoxication has been reported to reduce secretory epididymal components necessary for sperm maturation [reviewed in 2]. Mercury affects accessory sex gland function in rats and mice through androgen deficiency [4,5]. Popescu [6] described reproductive toxicity among workers occupationally exposed to mercury compounds. Keck et al. [7] reported elevated testicular mercury in an infertile man employed in the chloralkali industry.

Decrements in sperm count, motility, and morphology have been reported in methyl mercury exposed monkeys [8]. Vitamin E has a protective effect against mercury toxicity [9]. Vitamin E inhibits oxidative damage in the liver and other tissues caused by mercury and cadmium intoxication [9,10]. Although some data are available on the effects of inorganic mercury on fertility in experimental animals [11, 12], this study was undertaken to investigate the toxicity of mercury on reproductive organ function in male mice and to evaluate the preventive effect of vitamin E on mercuryinduced reproductive toxicity.

2. Materials and methods 2.1. Animals

* Corresponding author. Tel.: ⫹91-79-6302362; fax: ⫹91-796302654. E-mail address: [email protected] (M.V. Rao).

Healthy, adult male albino mice (Mus musculus) of Swiss strain, weighing from 35 to 40 g, were used for the

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experiments. Animals (80 to 90 days) were maintained on standard chow obtained from the National Institute of Occupational Health (NIOH; Government of India, Ahmedabad) without vitamin E supplementation, and water ad libitum. Animals were housed (60 to 100 cm2/animal in a cage) in an air conditioned animal house at a temperature of 26 ⫾ 2°C and exposed to 10 to 12 h of daylight. 2.2. Treatments Animals were divided into control and experimental groups. The experimental groups were given HgCl2 (1.25 mg/kg) daily for 45 days by gavage (0.2 ml/animal). To another group, HgCl2 was administered with vitamin E (2 mg/kg) for same period by the same route. A withdrawal group was treated with the same dose of HgCl2 for 45 days followed by a 45-day period of no treatment. The treatment duration of 45 days was selected as the length of the complete spermatogenic-epididymal maturation cycle in mice. The doses selected were based on previous work in our laboratory [13,14]. Animals were sacrificed after their respective treatments and their body weights were recorded together with individual organ weights. Testes, epididymides, and vasa deferentia were excised, blotted free of blood, and used for various biochemical studies. A sperm suspension from the cauda epididymis was made in normal saline (0.9% NaCl) and used for sperm parameters. 2.3. Sperm motility, count, and mating test A known amount of cauda epididymal tissue was teased in a desired volume of physiologic saline to release spermatozoa. Percent motility and count of cauda epididymal sperm were determined visually in a Neubauer chamber [15]. The units were expressed as percentage and million/ mL, respectively. A mating test was conducted by cohabiting estrous or proestrous female mice with treated males in a ratio of 2:1 according to the WHO protocol [16]. The next morning, vaginal smears were checked for the presence of sperm to confirm mating. This test was conducted before the end of each experimental period. The mating rate (%) was calculated as the number of sperm-positive females divided by the total number of males in the treatment group used for testing multiplied by 100. After confirmation of mating, the females were allowed to litter naturally to record litter size per animal. 2.4. Viability The ratio of live to dead spermatozoa was determined using 1% trypan blue as described in the method of Talbot and Chacon [17]. Briefly, undiluted sperm samples (0.2 ml) used for counting were incubated with 0.2 ml of 1% trypan blue stain for 15 min at 37°C. A drop of the suspension was

placed in a Neubauer chamber under a cover slip, allowed to settle for 1 min, and observed under a Nikon binocular microscope. The numbers of stained and unstained spermatozoa were scored in 10 to 20 separate fields. 2.5. Biochemical studies 2.5.1. Acid phosphatase (ACPase; E.C. 3.1.3.2) Enzyme activity in the testis was estimated by the method of Bessey et al. [18]. A known amount of tissue was homogenized in a desired volume of cold distilled water. To 0.2 ml of homogenate, 0.6 ml of substrate buffer (pH 4.8) was added. The blank contained 0.2 ml water instead of homogenate. The tubes were incubated for 30 min at 37°C following which 4 ml of NaOH was added to each tube. The resultant yellow colour was measured in a Spectronic-103 colorimeter and expressed as ␮mole p-nitrophenol/100 mg fresh tissue weight/30 min. 2.5.2. Alkaline phosphatase (ALKPase; E.C. 3.1.3.1) Enzyme activity in the testis was estimated by the method of Bessey et al. [18] using alkaline buffer (pH 10.5). The units were as for ACPase, above. 2.5.3. Succinate dehydrogenase (SDH; E.C. 1.3.9.9) The activity of SDH in the testis was assayed by the method of Beatty et al. [19]. To the sample tubes containing 0.4 ml of tissue homogenate in cold distilled water, 1 ml sodium succinate and 1 ml 2,4-iodophenyl-3,4-nitrophenyl5-phenyl tetrazolium chloride (INT) was added and incubated at 37°C for 15 min. In the blank, INT was replaced by 1 ml distilled water. The reaction was terminated by 0.1 ml of 30% trichloroacetic acid. The resulting formazan was extracted in 7 ml ethyl acetate and colour intensity was measured at 420 nm in a Spectronic 106 colorimeter. The activity was expressed as ␮g formazan/100 mg fresh tissue weight. 2.5.4. Cholesterol The estimation of testicular cholesterol was carried out by the method of Zlatki et al. [20]. To test tubes containing 5 ml of working FeCl3 solution, 0.2 ml of testis homogenate prepared in glacial acetic acid was added. The standard contained 0.1 ml of cholesterol solution (100 ␮g/0.1 ml). The contents were mixed and 3 ml of concentrated H2SO4 was added to all tubes. The optical density after colour development was read at 540 nm on a Spectronic 106 colorimeter and expressed as mg/100 mg tissue wt. 2.5.5. Protein The concentration of protein in all tissues was determined by the method of Lowry et al. [21] and expressed as mg protein/100 mg tissue. To 0.2 ml of tissue homogenate in distilled water, 0.6 ml of distilled water and 4.0 ml of alkaline CuSO4 solution were added, mixed, and incubated at room temperature for 15 min. Phenol reagent (0.4 ml)

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was added, mixed, and the preparation was incubated for 30 min at room temperature. The colour intensity was measured at 540 nm in a Spectronic 106 colorimeter. The same method was used for the estimation of protein in the cauda and caput epididymis and vas deferens. 2.5.6. Lipid peroxidation (LPO) Lipid peroxidation in all tissue was evaluated by the method of Ohkawa et al. [22]. To the sample tube containing 0.1 ml distilled water, 0.75 ml of 20% acetic acid, 0.2 ml sodium dodecyl sulphate, 0.75 ml of 1% thiobarbituric acid (TBA), and 0.1 ml of homogenate was added. To the blank tube, 0.1 ml distilled water was added instead of homogenate. The solutions were mixed and heated in a water bath for 60 min at 95°C. The tubes were cooled immediately and 2 ml of 10% trichloroacetic acid (TCA) was added, mixed, and centrifuged at 1000 g for 15 min. The O.D. of the supernatant was read at 532 nm. Lipid peroxidation was expressed as nmoles melonyldialdehyde (MDA) formed/ 100 mg tissue weight. 2.5.7. Glutathione (GSH) The concentration of glutathione in the testis was assayed by the method of Gru¨ nert and Philips [23]. A known amount of tissue was homogenized in 3 ml of 3% HPO3 and 1 ml distilled water and saturated with salt solution (1.5 g NaCl crystals). The solution was centrifuged at 800 g for 5 min. A 2-ml aliquot of supernatant was added to the sample tube containing 6 ml of saturated NaCl solution and allowed to stand for 10 min at 20°C. The blank tube was run with 2 ml of 2% HPO3 instead of the supernatant aliquot. Sodium nitroprusside and sodium cyanide, 1 ml each, was added to the blank and sample tubes respectively. The coloured complex developed was measured at 520 nm on a colorimeter against the blank tube. The units were ␮g glutathione/100 mg fresh tissue weight. 2.5.8. Ascorbic acid The level of ascorbic acid in the testis was estimated by the method of Roe and Kuether [24]. The tissue homogenate was prepared in 10 ml Norit reagent. The mixture was shaken well and allowed to stand for 15 min and then filtered through Whatman filter paper No.42. To 4 ml of homogenate, 1 ml of 2,4-dinitrophenyl hydrazine reagent was added and then a drop of 10% thiourea was mixed in order to activate the reaction. In the blank tube, 4 ml of 6% TCA was used instead of homogenate and in the standard tube, 4 ml ascorbic acid solution (10 ␮g/mL) was added. The contents of the tubes were mixed well, kept in boiling water for 15 min, and cooled in an ice bath. Then 5 ml of 85% sulphuric acid was added along the sides of the tubes. The tubes were allowed to stand for 30 min and the optical density was measured at 540 nm against the blank on a Spectronic 103 colorimeter. The concentration of ascorbic acid was expressed in mg/g tissue weight.

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2.5.9. Adenosine triphosphatase (ATPase; E.C. 3.6.1.3) The enzyme ATPase hydrolyzes the substrate ATP to adenosine diphosphate and inorganic phosphate, evaluated using the method of Quinn and White [25]. The inorganic phosphate was estimated by the method of Fiske and Subbarow [26] on a Spectronic 103 colorimeter at 660 nm and expressed as ␮mole inorganic phosphate released/h/100 mg tissue. The epididymal cauda epididymal tissue homogenate was prepared in cold distilled water. 2.5.10. Sialic acid Epididymal tissue sialic acid concentration was measured by the method of Jourdian et al. [27]. To 0.5 ml of homogenate prepared in distilled water, 0.1 ml of 0.6% resorcinol was added and boiled at 100°C for 15 min. The tubes were cooled and 1.25 ml of tertiary butyl alcohol was added. The tubes were kept at 37°C for 3 min for colour development and stabilization. The absorbance was determined on a Spectronic colorimeter at 630 nm. The sialic acid concentration was expressed as ␮g/mg tissue wt. 2.5.11. Phosphorylase (E.C. 2.4.1.1) The enzyme activity in vas deferens was estimated by the modified technique of Cori et al. [28] and phosphate estimation was performed by the method of Fiske and Subbarow [26]. A known amount of tissue was homogenized and 0.1 ml of this homogenate was added to a cold-incubated mixture containing 0.2 ml sodium nitrate buffer (0.1 M, pH 5.9) and 0.3 ml potassium fluoride (0.15 M). The solutions were incubated for 15 min at 37°C after which 1 ml of 10% TCA was added to stop the enzyme activity. The incubated solution was filtered and the filter paper was washed repeatedly with a small amount of water at a time to make the filtrate volume up to approximately 8 ml in tubes placed in an ice cold water bath at 20°C. A control tube was prepared using 0.5 ml of TCA in place of the homogenate. Ammonium molybdate, 1 ml, was added to the tubes followed by 0.5 ml of 2,4-aminonaphthol sulphonic acid (ANSA) reagent to make the total volume up to 10 ml. The solution was mixed and allowed to stand for 6 min and the optical density of the developed colour was read at 660 nm. The enzyme activity was expressed as ␮moles inorganic phosphate released/100 mg fresh tissue weight/15 min. 2.5.12. Glutathione peroxidase (GSH-Px; E.C. 1.11.1.9) The testicular enzyme activity was assayed by the modified technique of Paglia and Valentine [29]. A known tissue weight was homogenized in the required volume of 0.01% digitonin and centrifuged at 12,000 g for 30 min at 4°C. This supernatant (0.1 ml) was used in the reaction mixture of 0.8 ml. The reaction was initiated by addition of 0.1 ml H2O2. The decrease in absorbance at 340 nm was recorded for 3 min at an interval of 1 min to calculate enzyme activity.

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Table 1 Body weight (g) and organ weights (mg) of control and experimental animals Parameters

Body weight (g) Testis Caput epididymidis Cauda epididymidis Vas deferens

Control

38.8 ⫾ 1.6 123.5 ⫾ 4.2 27.8 ⫾ 0.7 19.4 ⫾ 0.7 14.0 ⫾ 0.3

45 d treatments Vitamin E

HgCl2

HgCl2 ⫹ Vitamin E

37.0 ⫾ 1.2 130.5 ⫾ 3.2 29.0 ⫾ 0.6 20.5 ⫾ 0.6 16.0 ⫾ 0.8

43.3 ⫾ 1.2 97.5 ⫾ 6.2a 32.4 ⫾ 2.1 12.3 ⫾ 1.3a 9.1 ⫾ 0.2

40.2 ⫾ 1.2 122.6 ⫾ 2.6 28.5 ⫾ 1.6 17.1 ⫾ 1.3 12.3 ⫾ 0.6

45-day HgCl2 followed by 45-day withdrawal 40.1 ⫾ 1.0 114.4 ⫾ 3.7b 28.4 ⫾ 1.2 17.2 ⫾ 0.6 11.1 ⫾ 0.2

Values are mean ⫾ SE, n ⫽ 10 animals per group. a,b Significantly different from the other treatment groups by ANOVA and Scheffe’s test.

2.5.13. Glycogen The concentration of glycogen in the vas deferens was estimated using the method of Seifter et al. [30]. A known amount of tissue was digested in 2 ml of 30% KOH solution followed by washing with ethanol. The digest was extracted in ethanol, precipitated at 4°C, and dissolved in a known volume of distilled water. A 1 ml aliquot of this sample was added to 4 ml anthrone reagent and kept in a boiling water bath for 4 min. After cooling, the resultant colour was read at 620 nm wavelength on a Spectronic 103 colorimeter and expressed as ␮g/100 mg tissue weight.

photometry. The recovery percentage was 94 –95% and levels were expressed as ng/mg tissue weight.

2.5.14. Radioimmunoassay (RIA) for serum testosterone Blood was collected by cardiac puncture at the time of death. The serum was separated by centrifugation and was used for assaying androgen using RIA kits (Amar Diagnostics, Mumbai) based on the method of Castro et al. [31]. Antibody produced against testosterone in rabbits was used. The cross reaction with dihydrotestosterone (DHT) was 16 –17% and the units were ng/mL serum.

3. Results

2.6. Statistics For all biochemical estimations, a minimum of 10 to 12 replicates were used for each parameter and tissue. The data were statistically analysed using ANOVA followed by Scheffe’s test for multiple pairwise comparisons [33]. A significant level of P ⬍ 0.05 was accepted.

3.1. Body weights The body weights of the HgCl2 treated mice did not differ significantly compared to the other groups of animals (Table 1). 3.2. Organ weights

2.5.15. Mercury levels The acid digestion method [32] was used with a known amount of tissue (200 –500 g). The digested sample was diluted to the required volume with appropriate reference materials as quality controls. These samples were analyzed by making use of cold vapour atomic absorption spectro-

No significant differences were found in weights of caput epididymidis and vas deferens after HgCl2 treatment for 45 days. However, testis, vas deferens, and cauda epididymal weights were significantly altered by mercury treatment. With vitamin E coadministration, these values were com-

Table 2 Sperm parameters, mating rate, and serum testosterone levels of control and experimental animals Parameters

Sperm motility (%) Sperm count (million/mL) Viable sperm (%) Mating rate (%) Litter size/animal Serum testosterone (ng/mL)

Control

45-day treatments

45-day HgCl2 followed by 45-day withdrawal

Vitamin E

HgCl2

HgCl2 ⫹ Vitamin E

75.90 ⫾ 1.97 42.50 ⫾ 1.91

75.00 ⫾ 1.80 48.50 ⫾ 1.1

38.57 ⫾ 2.39a 15.64 ⫾ 1.18a

73.91 ⫾ 0.548 38.07 ⫾ 1.19

55.85 ⫾ 2.65b 33.50 ⫾ 0.92b

63.33 ⫾ 2.61 92.50 ⫾ 1.51 11.00 ⫾ 0.60 4.21 ⫾ 0.19

72.00 ⫾ 1.61 96.50 ⫾ 1.31 10.00 ⫾ 0.70 4.50 ⫾ 0.14

39.00 ⫾ 2.31a 0 ⫾ 0.00a 0 ⫾ 0.00a 1.64 ⫾ 0.18a

62.00 ⫾ 0.92 87.50 ⫾ 0.81 9.50 ⫾ 0.61 3.23 ⫾ 0.02

58.57 ⫾ 1.93 50.00 ⫾ 0.60b 6.50 ⫾ 0.35b 2.20 ⫾ 0.05b

Values are Mean ⫾ SE; n ⫽ 10 animals per group. a,b Significantly different from other groups by ANOVA and Scheffe’s test.

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Table 3 Testicular biochemical parameters Parameter

Control

45-day treatments HgCl2 ⫹ vitamin E

HgCl2 Acid phosphatase (␮moles p-nitrophenol released/100 mg/30 min) Alkaline phosphatase (␮moles pnitrophenol released/100 mg/30 min) Succinate dehydrogenase (␮g formazan formed/15 min/100 mg) Cholesterol (mg/100 mg) Protein (mg/100 mg tissue wt.) Lipid peroxidation (nmoles MDA formed/100 mg) Glutathione Peroxidase (mU/mg/min) Glutathione (␮g/100 mg) Ascorbic Acid (mg/g tissue wt.)

45-day HgCl2 followed by 45-day withdrawal

12.44 ⫾ 4.37

6.40 ⫾ 0.59

a

10.90 ⫾ 0.20

9.79 ⫾ 0.56

11.13 ⫾ 0.38

4.61 ⫾ 0.34a

10.79 ⫾ 0.29

8.92 ⫾ 0.24

465 ⫾ 12.13

233 ⫾ 19.33a

425 ⫾ 12.80

410 ⫾ 6.51

0.285 ⫾ 0.006 15.03 ⫾ 0.35 14.33 ⫾ 0.279

0.140 ⫾ 0.001a 9.27 ⫾ 0.67a 29.63 ⫾ 0.23a

0.247 ⫾ 0.005 15.43 ⫾ 0.23 15.57 ⫾ 0.21

0.212 ⫾ 0.001 10.11 ⫾ 0.56 22.32 ⫾ 0.25b

0.036 ⫾ 0.01 101.45 ⫾ 2.68 1.61 ⫾ 0.036

0.01 ⫾ 0.001a 56.28 ⫾ 3.06a 0.885 ⫾ 0.018a

0.039 ⫾ 0.004 98.87 ⫾ 1.86 1.545 ⫾ 0.013

0.018 ⫾ 0.00b 90.08 ⫾ 1.74 0.819 ⫾ 1.70b

Values are Mean ⫾ SE; n ⫽ 10 animals per group. a Significantly different from other groups by ANOVA and Scheffe’s test. b Significantly different from control and HgCl2 ⫹ vitamin E groups by ANOVA and Scheffe’s test.

parable to those in control mice. In the withdrawal group of mice, testis weight partially recovered. Vitamin E alone had no effect (Table 1). 3.3. Sperm motility, count, viability, and mating rate (Table 2) Sperm count and motility from the cauda epididymis was markedly reduced by HgCl2, but was comparable to normal levels when vitamin E was coadministered with HgCl2. The mating rate was zero in HgCl2-treated mice. Coadministration of vitamin E with HgCl2 was associated with a mating rate of 85–90% compared to 90 –95% in controls. Similar findings were obtained with respect to litter size. The frequency of nonviable sperm increased markedly after HgCl2 treatment with no significant effect associated with vitamin E cotreatment. Animals receiving vitamin E alone had sperm parameters similar to control values. Mercury intoxicated mice had a significant decline in serum testosterone levels. These levels in HgCl2 ⫹ vitamin E treated animals were comparable to the control values. The withdrawal group had a partial recovery in testosterone levels.

3.4. Biochemical parameters 3.4.1. Testis (Table 3) HgCl2 treatment for 45 days was associated with a significant reduction in protein content compared to other groups. The vitamin E supplemented and withdrawal groups had values comparable to control levels. A significant decline in ACPase, ALKPase, SDH, and GSH-Px activities were observed in the HgCl2 group. A significant increase in LPO levels was noted after HgCl2 treatment. These levels did not differ from control levels when vitamin E was coadministered with HgCl2. Mercury treatment also resulted in a reduction in glutathione and ascorbic acid levels. Coadministration of vitamin E with HgCl2 resulted in maintenance of glutathione and ascorbic acid at control levels. Withdrawal of HgCl2 treatment for 45 days resulted in partial recovery of these parameters. 3.4.2. Epididymis (Table 4) Caput and cauda epididymal proteins, sialic acid, and ATPase levels were significantly reduced by HgCl2 treatment with preservation at control levels in the HgCl2 ⫹

Table 4 Cauda and caput epididymal biochemical parameters Parameter

Cauda ATPase (␮moles ip/100 mg) Protein (mg/100 mg) Sialic acid (␮g/mg) Caput Protein (mg/100 mg) Sialic acid (␮g/mg)

Control

45-day treatments

45-day HgCl2 followed by 45-day withdrawal

HgCl2

HgCl2 ⫹ vitamin E

0.50 ⫾ 0.01 17.70 ⫾ 0.85 4.45 ⫾ 0.27

0.278 ⫾ 0.02a 7.65 ⫾ 0.35a 1.25 ⫾ 0.25a

0.489 ⫾ 0.023 16.00 ⫾ 0.46 4.40 ⫾ 0.26

0.407 ⫾ 0.01 14.24 ⫾ 0.60b 3.31 ⫾ 0.35b

16.57 ⫾ 0.21 5.05 ⫾ 0.25

10.65 ⫾ 0.50a 1.77 ⫾ 0.38a

14.25 ⫾ 0.250 4.01 ⫾ 0.20

11.36 ⫾ 0.34b 3.95 ⫾ 1.21

Values are Mean ⫾ SE; n ⫽ 10 animals per group. a Significantly different from other groups by ANOVA and Scheffe’s test. b Significantly different from control and HgCl2 ⫹ vitamin E groups by ANOVA and Scheffe’s test.

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Table 5 Vas deferens biochemical parameters Parameter

Control

45-day treatments HgCl2 ⫹ vitamin E

HgCl2 Glycogen (␮g/100 mg) Phosphorylase (mg/100 mg) Protein (mg/100 mg)

466 ⫾ 9.36 5.27 ⫾ 0.11 15.50 ⫾ 0.63

710 ⫾ 12.60 2.44 ⫾ 0.05a 9.60 ⫾ 0.43a

a

493 ⫾ 10.62 4.33 ⫾ 0.056 15.43 ⫾ 0.602

45-day HgCl2 followed by 45-day withdrawal 674 ⫾ 16.60b 3.89 ⫾ 0.02b 12.03 ⫾ 0.55

Values are Mean ⫾ SE; n ⫽ 10 animals per group. a Significantly different from other groups by ANOVA and Scheffe’s test. b Significantly different from control and HgCl2 ⫹ vitamin E groups by ANOVA and Scheffe’s test.

vitamin E group. The withdrawal groups showed partial recovery with respect to these parameters. 3.4.3. Vas deferens (Table 5) Glycogen levels were significantly elevated and phosphorylase activity decreased in the HgCl2-treated group. There were no differences from control levels after treatment with HgCl2 ⫹ vitamin E. Total proteins were decreased by HgCl2 administration; this decrease was prevented by coadministration of vitamin E. The vas deferens biochemical changes showed partial recovery in the withdrawal group. 3.4.4. Mercury levels (Table 6) Mercury levels in the testis, epididymis, and vas deferens exhibited a significant increase after 45 days of mercury treatment. These tissues in HgCl2 ⫹ vitamin E treated mice had lower concentrations of mercury compared to those of HgCl2-treated mice. The withdrawal group showed a significant decrease in mercury accumulation only in the caput epididymis. 3.5. Vitamin E effect Body and reproductive weights, sperm parameters, fertility, and serum testosterone were not altered by treatment with vitamin E alone (Tables 1 and 2).

4. Discussion Oral administration of HgCl2 for 45 days to adult male mice brought about no significant change in body weight.

There were decreases in testis, vas deferens, and cauda epididymis weight. A reduction in sperm count revealed an inhibitory effect of HgCl2 on spermatogenesis in parallel to a reduction in testis weight. Adverse effects of HgCl2 on mammalian testicular tissue have been reported with marked testicular spermatogenic degeneration at the spermatocyte level in rats [34]. Rao [2] also mentioned suppression of testicular function in mice treated with inorganic and methyl mercury, in support of our present data. It is known that mercury passes through the blood-testis barrier to induce testicular damage [11,35]. Suppression of sperm motility by mercury has been reported in different mammalian species in association with altered sperm metabolism and morphology [2,36]. Similar observations have been made in the sperm of men who were occupationally exposed to mercury [6]. Mercury interferes with cell membrane structure and also immobilizes sperm by inhibition of microtubule assembly [36 –38]. The reduction in viability of sperm in our study is consistent with such effects. Testicular SDH, ALKPase, ACPase, protein, ascorbic acid, GSH, GSH-Px, and lipid peroxidation (LPO) levels exhibited alterations after mercury treatment. A depletion of testicular GSH in mercury treated animals has been reported by other investigators [39,40]. The cysteinyl residue of glutathione provides thiols, which are important for the detoxification of metabolically generated oxidizing agents. These effects are magnified by LPO and reduced GSH-Px levels. Epididymal physiology in treated mice may have been disrupted as evidenced by alterations in epididymal protein, ATPase, and sialic acids. Epididymal sialioproteins and other proteins are important for sperm maturation. Mercury ions are known to enter the epididymis through the blood-

Table 6 Mercury levels (␮g/mg tissue) in reproductive organs Organ

Testis Caput epididymidis Cauda epididymidis Vas deferens

Control

1.57 ⫾ 0.11 0.72 ⫾ 0.29 0.58 ⫾ 0.12 1.66 ⫾ 0.13

45-day treatments HgCl2

HgCl2 ⫹ vitamin E

3.12 ⫾ 0.19a 2.62 ⫾ 0.16a 1.94 ⫾ 0.80a 2.81 ⫾ 0.00a

0.66 ⫾ 0.09 0.94 ⫾ 0.14 0.43 ⫾ 0.03 0.82 ⫾ 0.16

45-day HgCl2 followed by 45-day withdrawal 1.34 ⫾ 0.04 0.59 ⫾ 0.04b 0.54 ⫾ 0.17 1.58 ⫾ 0.02

Values are Mean ⫾ SE; n ⫽ 12 animals per group. a Significantly different from other groups by ANOVA and Scheffe’s test. b Significantly different from control and HgCl2 ⫹ vitamin E groups by ANOVA and Scheffe’s test.

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epididymal barrier [35,41]. Sharma [42] also reported morphologic defects in the epididymis associated with mercury treatment. In the vas deferens, glycogen levels were increased and phosphorylase levels were decreased after HgCl2 treatment, suggesting an inhibitory action on carbohydrate metabolism. These findings might reflect an altered ability to support the viability of sperm stored in this organ. The reduced serum testosterone levels support the possibility of reproductive tract alterations due to androgen deficiency following mercury treatment. The accumulation of testicular cholesterol, a precursor for steroidogenesis, is consistent with an impairment of androgen synthesis. Mating success was nil after HgCl2 treatment. The lack of sperm-positive vaginal smears could have been due to inhibition of mounting, intromission, or ejaculation. When mice were permitted to recover for 45 days after withdrawal of HgCl2 treatment, partial recovery in most of the parameters was evident as the sperm-positive rate reversed from 0 to 87.5%. Cotreatment with HgCl2 and vitamin E was associated with a control rate of sperm positivity, perhaps due to protection by vitamin E from oxidative tissue damage induced by mercury ion [43,44]. Vitamin E prevents lipid peroxidation and maintains GSH and ascorbic acid levels in damaged tissue by inhibiting free radicalsformation [45,46]. Vitamin E also can inhibit conversion of -SH to SS groups, thereby maintaining protein function [43,47]. It is possible, however, that coadministration of HgCl2 and vitamin E was protective due to impaired absorption of mercury in the presence of vitamin E in the gastrointestinal tract. We have not located information in the published literature on mercury absorption in the presence of vitamin E, but the lower testicular and epididymal mercury levels in cotreated animals are consistent with this possibility. In summary, it is likely that HgCl2 exerts adverse effects on the testis, vas deferens, and epididymis by inducing androgen deficiency and perhaps through direct toxic effects on these tissues. These changes were not observed with coadministration of vitamin E with HgCl2.

[8]

[9]

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