chelation therapy and vanadium: effect on reproductive

Indian Journal of Experimental Biology Vol. 45, June 2007, pp. 515-523

Chelation therapy and vanadium: Effect on reproductive organs in rats

Sadhana Shrivastava, Anjana Jadon, Sangeeta Shukla* & Ramesh Mathur

Reproductive Biology and Toxicology Laboratory, School of Studies in Zoology, Jiwaji University, Gwalior 474 011, India

Received 12 July 2006; revised 23 January 2007

Present investigation was planned to evaluate the therapeutic effectiveness of chelating agents against vanadium intoxication on blood and reproductive organs of rats. Male and female albino rats were injected vanadyl sulphate (7.5 mg/kg, po, for 21 days, 5 days in a week). Chelating agents tiron (T) alone and in combination with lipoic acid (LA), vitamin E (vit E) and selenium (Se) were given for 2 days/week. With the administration of vanadyl sulphate to rats fructose level in seminal vesicles was significantly (P<0.05) declined. The activities of alkaline phosphatase and adenosine triphosphatase were also decreased, whereas glycogen content and acid phosphatase activity increased in testis, seminal vesicles, ovaries and uterus after toxicant exposure. Significant changes in serum transaminases, serum alkaline phosphatase and lactate dehydrogenase were recouped by chelation therapy. Lipid peroxidation, reduced glutathione level and triglycerides levels altered significantly after exposure to vanadium in rats. The ultrastructural damage in spermatogenic stages in treated animals showed recovery pattern after therapy. Co-treatment with antioxidants restored these activities. The most effective combination was tiron + selenium followed by tiron + vitamin E, and tiron + lipoic acid.

Keywords: Lipoic acid, Selenium, Tiron, Vanadyl sulphate, Vitamin E.

Vanadium (VOSO4) is an important environmental and industrial pollutant. It is a dietary micronutrient and it has been recently considered as a pharmaco-logical agent. Its compounds have been associated with various implications in the pathogenesis of human diseases such as bronchial asthma, pneumonia and tightness of the chest1. Vanadium is capable of significantly decreasing the percentage of gamma-tubulin in all analysed testicular cells (Sertoli, Leydig and germ cells)2,3. Embryotoxicity, fetotoxicity and teratogenecity have been reported in rats, mice and hamsters after vanadium exposure4. The mechanism involved in its toxicity is still unclear5,6. Tiron (4,5-dihydroxy-1,3-benzene disulphonic acid disodium salt) is a phenolic compound used as a chelating agent in this experiment. Studies in reproductive organs are limited, so the present work was planned to identify the effect of tiron along with lipoic acid, vitamin E and selenium against vanadium toxicity in male and female reproductive organs.

Materials and Methods Sprague Dawley female/male albino rats weighing 160 ± 10 g from animal facility of the department were selected. They received a standard pellet diet (Pranav Agro Industries, New Delhi, India) having metal contents in ppm dry weight Cu, 10; Mn, 33; Zn, 45; and Co, 5). Drinking water was also provided ad libitum. Animals used in this study were treated and cared for in accordance with the guidelines recommended by the Committee for the Purpose of Control and Supervision of Experiments on Animals (CPCSEA), Government of India, Ministry of Culture, Chennai. The dose of the chelating agent was prepared daily and its pH was adjusted to 6.4 with sodium bicarbonate before administration. The selected animals were divided into 6 groups. Group 1, normal saline (4 ml/kg, po); group 2, VOSO4 (7.5 mg/kg, po 5 days in a week for 21days); group 3, VOSO4 followed by tiron (606 mg/kg, ip 2 days 6th-7th

day/week); group 4, VOSO4 + tiron + lipoic acid (30 mg/kg, ip); group 5, VOSO4 + tiron + vitamin E (50 mg/kg, po); and group 6, VOSO4 + tiron + selenium (0.5 mg/kg, po) of 5 animals in each group.

Animals were sacrificed under light ether anaesthesia after 24 hr of last treatment. The testis, epididymidis, seminal vesicles, uterus, ovary and liver

________________ *Correspondent author Phone: 0751-4016750 Fax No.: 0751-2341450 E-mail: [email protected]; [email protected]

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were collected for different assays. Serum trans-aminases (AST and ALT)7, lactate dehydrogenases (LDH)8 and triglycerides (TG) were estimated using the kit purchased from MERK, Ltd. Blood was collected directly from the heart by puncturing the retro-orbital venous sinus9, standard techniques were used to determine glycogen content in fresh tissue10. Homogenate in isotonic solution was processed for determination of total proteins11, activities of acid (ACPase) and alkaline phosphatases (ALPase)12, adenosine triphosphatase (ATPase)13, fructose14, hepatic lipid peroxidation (LPO)15 and reduced glutathione (GSH)16.

Histopathology—Testis, epididymis, seminal vesicles, uterus and ovary were fixed for examination under light microscope. For electron microscopy, samples were fixed in modified Karnovsky’s fluid and post-fixed for 2 hr in 1% osmium tetraoxide at 4°C. Ultrathin sections (60-80 nm thick) were cut using an ultracut E (Reichert Jung) Ultramicrotome. The sections were stained in alcoholic uranyl acetate (10 min) and lead acetate (10 min) before examining the grides in a transmission electron microscope (Philips, CM-10) operated at 60-80 Kv17.

Statistical analysis—Significance of difference among various groups was calculated by one way analysis of variance (ANOVA) followed by Student’s t test. P value was taken as significant at 5% level18.

Results

Vanadium administration produced severe alterations in various blood/serum biochemical variables and in blood cellular components. One of the most sensitive and dramatic indicators of hepato-cyte injury is the release of intracellular enzymes, such as transaminases and lactate dehydrogenase in

the circulation. Vanadium induced significant eleva-tion in the activities of serum AST, ALT and LDH whereas, triglyceride contents were decreased significantly. In this study, the altered level of these parameters were significantly restored with the therapy of V+T+Se whereas other combination were not effective at this level (Table 1). A significant increase in hepatic lipid peroxidation level was observed whereas, significant decrease was evaluated in hepatic reduced glutathione and fructose in seminal vesicles after vanadium intoxication. Treatment with different combinations reversed these biochemical parameters significantly (Table 2). Increase level of glycogen contents was observed after toxicity in uterus, ovary, testis and seminal vesicles (Tables 3, 4). Protein contents of these organs were also altered after subchronic exposure of vanadium. Significant recoupment was seen with tiron and selenium combination. Vulnerable effect of vanadium was seen on the acid and alkaline phosphatase activities (Tables 5, 6). The present results demonstrated that activity of acid phosphatase increased significantly, on the contrary alkaline phosphatase activity was inhibited significantly in all these organs. Treatment with therapeutic agents restored these enzymatic activities. Tiron along with selenium showed maximum recovery in both the parameters. Variation between all the groups were performed with one way ANOVA at 5% level. Adenosine triphosphatase showed decreased values when compared to the controls (Table 7). Treatment with tiron with and without vitamin E, Se, lipoic acid showed improvement in all the reproductive organs, however T+ Se showed maximum recoupment.

Male reproductive organs—The ultrastructure of testis showed damage in various stages of spermato-

Table 1—Effect of chelating agents on blood biochemical parameters against vanadium.

(Values are expressed as mean ± SE of 5 animals) Treatments AST ALT LDH Triglycerides

(IU/l) (IU/l) (µ mole/min/l) (mg/dl)

N 68.1 ± 4.39 54.0 ± 3.16 40.4 ± 3.11 9.60 ± 0.63 V 94.9 ± 4.76# 148.1 ± 9.67# 87.6 ± 6.90# 6.19 ± 0.31#

V + T 73.2 ± 3.87 * 83.6 ± 5.54* 55.6 ± 3.70* 8.72 ± 0.43* V + T + LA 84.1 ± 3.95* 105.2 ± 9.61* 72.2 ± 4.36 8.86 ± 0.57*

V + T + vit E 79.2 ± 5.41* 93.6 ± 6.53* 63.6 ± 4.43* 7.49 ± 0.45* V + T + Se 70.4 ± 5.40* 69.2 ± 5.16* 45.6 ± 3.29* 9.52 ± 0.47* ANOVA 6.52@ 27.17@ 19.02@ 8.99@

# P ≤ 0.05 when compared with control group, *P ≤ 0.05 when compared with V treated group. ANOVA (F values at 5% level) @ Significant, ns Not significant.

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genesis. The testis showed apoptotic germ cells, cytoplasmic vacuolation, deformities of head, nucleus and acrosome of spermatozoa, damaged Leydig cells were noted in vanadium treated animals. Damaged

Sertoli cells were loosely attached with basal membrane. The spermatogonia have deformed nuclei (Fig. 1). Profuse distribution of lipid droplet was seen in cytoplasm of Sertoli cells and degenerated

Table 2—Effect of chelating agents on tissue biochemical parameters against vanadium.

(Values are expressed as mean ± SE of 5 animals) Treatments LPO GSH Fructose:Seminal vesicles

(n mole MDA /mg protein) (µ mole/g) (µg/100 mg tissue)

N 0.38±0.01 8.80±0.33 336.8±21.6 V 0.70±0.04# 6.32±0.24# 300.4±16.1#

V+T 0.41±0.02* 7.79±0.47* 334.4±18.9* V+T+LA 0.45±0.04* 7.34±0.38 335.4±19.6* V+T+vitE 0.42±0.03* 7.79±0.47* 337.6±21.1* V+T+Se 0.39±0.02* 8.28±0.60* 335.2±18.6* ANOVA 17.18@ 4.73@ 0.49ns


Table 3—Effect of chelating agents on glycogen against vanadium

(Values are expressed as mean ± SE of 5 animals) Glycogen (mg/100 g)

Treatments Uterus Ovary Testis Seminal vesicles

N 27.6 ± 1.92 69.4 ± 3.89 68.44 ± 3.58 60.41 ± 3.10 V 61.6 ± 4.96# 122.8 ± 8.61# 97.97 ± 4.33# 90.49 ± 4.72#

V + T 33.8 ± 2.55 * 72.1 ± 5.10* 73.78 ± 3.74* 70.52 ± 76.2* V + T + LA 38.2 ± 5.57* 99.6 ± 8.92 74.73 ± 3.55* 76.23 ± 3.62* V + T + vitE 41.6 ± 3.56* 103.2 ± 7.89 61.71 ± 2.70* 80.95 ± 4.78 V + T + Se 28.2 ± 1.34* 67.6 ± 4.32* 65.65 ± 3.32* 63.24 ± 3.43* ANOVA 14.03@ 19.04@ 16.17@ 9.84@


Table 4—Effect of chelating agents on proteins against vanadium induced toxicity in rats

(Values are expressed as mean ± SE of 5 animals) Treatments Proteins (mg/100 mg)

Uterus Testis Seminal vesicles

N 12.5 ± 0.83 11.5 ± 0.61 15.3 ± 0.81 V 8.8 ± 0.59# 17.1 ± 0.90# 25.3 ± 1.81#

V + T 10.3 ± 0.83* 12.8 ± 0.67* 15.5 ± 1.14 V + T + LA 8.8 ± 0.73 13.0 ± 0.70* 16.9 ± 1.10

V + T + vit E 10.4 ± 0.89* 13.4 ± 0.66* 14.0 ± 0.98 V + T + Se 11.3 ± 1.07* 13.4 ± 0.65* 14.6 ± 0.79 ANOVA 4.18@ 8.93@ 16.23@


Table 5—Effect of chelating agents on acid phosphatase against vanadium

(Values are expressed as mean ± SE of 5 animals) Acid phosphatase (mg Pi/100 g/h)

Treatments Uterus Testis Seminal vesicles

N 246.6 ± 13.9 261.0 ± 13.2 104.3 ± 6.83 V 355.4 ± 18.2# 351.4 ± 26.4# 187.3 ± 11.5

V + T 229.6 ± 9.6* 310.3 ± 17.4* 128.6 ± 6.78 V + T + LA 201.6 ± 13.8* 224.7 ± 13.4* 103.7 ± 6.21

V + T + vit E 228.0 ± 12.3* 328.1 ± 16.2* 116.7 ± 7.85 V + T + Se 241.2 ± 21.8** 317.4 ± 17.4* 112.5 ± 6.58 ANOVA 16.15@ 8.86@ 20.23@



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spermatids were noted (Fig. 2). In light microscopical observations, the testis showed wavy tunica albuginea and loosely arranged central seminiferous tubules due to the shrinkage of Leydig cells. The spermatozoa were scanty (Fig. 3). The therapy of tiron maintained the germinal epithelium, Leydig cells, Sertoli cells and thick fibrous basement membrane however, tiron along with Se showed significant improvement. Lumen was filled with spermatozoa (Fig. 4). Toxicant caused irregular shape ductules, less stereocilia, apical nuclei and reduced number of sperms in caput and cauda epididymidis (Figs 5, 7). Conjoint treatment of T + Se, improved epithelial height of ductules with ciliary arrangement and number of sperms (Figs 6, 8). Vanadium exposure reduced the connective tissue stroma and secretion of seminal vesicles so, lumen

was clear (Fig. 9). Therapy of tiron showed normal epithelium height, well maintained basement membrane, normal secretion and musculature. Therapy with T + Se showed normal appearance of the epithelial cells and musculature, but secretion was reduced (Fig. 10).

Female reproductive organs—Vanadium caused fibrosis in stroma, disorganized uterine glands and disruption of columnar epithelium. Some debris was observed in the lumen (Fig. 11). With the treatment of tiron endometrium of uterus was intact with normal columnar epithelial cells. Stroma was well maintained and vascularity was prominent. With conjoint treat-ment of T+LA uterus showed considerable improve-ment in the endometrium and myometrium. Treatment of T+Se showed intact columnar epithelium with wide and clear lumen. The uterine glands showed normal picture and stromal cells were clear (Fig. 12). After vanadium exposure corpora lutea showed hypertrophy and vacuolation with darkly stained nuclei. Graffian follicles and ova showed degeneration (Fig. 13). Tiron + Se treatment showed well formed follicles. Graffian follicle also showed recovery (Fig. 14).

Discussion Vanadium is mainly present in the blood plasma,

where it is bound with transferrin19. In the present study, activities of serum transaminases (AST and ALT) increased significantly after vanadium exposure which might be due to tissue lysosomal disruption, phagocytosis and acute cellular injury20. Lactate dehydrogenase is an intracellular enzyme and a sole marker of liver injury. These enzymatic activities re-couped significantly with the treatment of T/T+LA/ T+vitE/T+Se. It is involved in the inter conversion of lactate to pyruvate. In this study, it increased after toxicant administration. Vanadate may have some interaction with the enzyme moiety and or coenzyme system21. In the present study, triglycerides level lowered significantly after 21 days administration of vanadium. The cytoplasm of the cell is full of lipid vacuoles that are almost exclusively triglycerides and serves as an energy reservoir. Vanadate decreased the triglycerides and cholesterol levels in diabetic animals indicating the insulin mimic effect of vanadium20.

A significant rise was seen in lipid peroxidation, it is supported by various authors22. Vanadium administration decreased the hepatic GSH level. Its accumulation in the tissues with its concomitant redox cycling may lead to lipid peroxidation, which

Table 6—Effect of chelating agents on alkaline phosphatase against vanadium.

(Values are expressed as mean ± SE of 5 animals) Alkaline phosphatase (mg Pi/100 g/h)


N 394.3 ± 28.8 347.4 ± 20.7 491.2 ± 25.7 V 181.8 ± 12.1# 196.2 ± 11.3# 289.8 ± 21.9

V + T 342.7 ± 18.5* 302.1 ± 23.1* 420.7 ± 22.1 V + T + LA 274.8 ± 17.6* 283.4 ± 20.9* 362.8 ± 21.9

V + T + vit E 296.8 ± 24.3* 256.7 ± 12.1* 472.8 ± 27.8 V + T + Se 361.4 ± 20.6* 358.6 ± 23.0* 387.7 ± 26.4* ANOVA 16.15@ 12.25@ 11.50@

# P ≤ 0.05 when compared with control group, *P ≤ 0.05 when compared with V treated group. ANOVA (F values at 5% level) @Significant, nsNot significant.

Table 7—Effect of chelating agents on adenosine triphosphatase against vanadium.

(Values are expressed as mean ± SE of 5 animals) Adenosine triphosphatase (mg Pi/100 g/min)


N 849.4 ± 49.9 1381.6 ± 95.3 894.8 ± 62.0 V 409.4 ± 31.7# 950.8 ± 93.1# 656.8 ± 41.7# T 768.2 ± 42.6* 1306.4 ± 98.3* 804.4 ± 67.7

V + T 756.2 ± 41.4* 1211.4 ± 76.5 742.8 ± 44.1 V + T + LA 750.4 ± 36.9* 1257.2 ± 91.2 756.4 ± 39.1

V + T + vit E 796.8 ± 40.5* 1380.8 ± 88.9* 871.2 ± 48.1* ANOVA 18.44@ 4.47@ 4.05@



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Fig. 1—Spermatogonia and Sertoli cells were attached to the basal membrane (× 850). Fig. 2—Sertoli cell was clearly seen along with many droplets of varying sizes around the nucleus (× 850).


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consequently damage biological membranes and lysosomal enzymes23. This pro-oxidant effect of vanadium is evident in our studies by elevation of LPO and depletion of GSH24. It may be due to its consumption of available GSH in the formation of vanadyle/GSH complex. A constant supply of glucose is considered to be an essential requirement for the proper functioning of the testes and malnutrition of germ cells25. Vanadium administration for 21 days

increased glycogen content in testis, seminal vesicle, uterus and ovary. It may be due to depression of the activity of 3-phosphoglycerate dehydrogenase and resulting in reduced glycolysis rate26. Depletion of protein content corresponds to the inhibition of spermatogenesis and suppressed Leydig cell function. Se prevented induction of LPO maximally as it is an important element in glutathione peroxidase that plays a significant role in protecting tissues by catalyzing

Figs 3-8—[3—Vanadium induced degeneration in Leydig cells along with spermatogonia. 4—Therapy with T + Se, showed spermatozoa in lumen. 5—Vanadium administration showed irregular shape ductules of caput epididymidis.

6—Therapy with T+Se showed maintain ciliary arrangement and basal nuclei in the epithelium cells of ductule. 7—Vanadium caused number of spermatozoa in lumen of cauda epididymidis 8—Combination therapy of T + Se

showed sperms in the lumen of rat testis (Figs 3-8, × 120)]


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peroxides which are generated in both metabolic processes and the environment.

Testicular acid phosphatase is localized in the Sertoli cells and the Leydig cells and is an accurate marker of specific stages of spermatogenesis and activity correlates with the activity of testosterone. In the present observation, increase acid phosphatase activity was seen in the reproductive system which may be due to severe impairment in the reproductive

system. Significant decrease was seen in alkaline phosphatase and adenosine triphosphatase. Vanadate (VO3) interferes with a large variety of phosphate dependent enzymes27. It is a potent inhibitor of membrane bound ATPase28. Vanadate can be bioequivalent to phosphate and replace it in cellular metabolism29. Na metavanadate decreased testicular sperm counts and the epididymis weight. Chronic exposure of vanadium reduces pregnancy rate,

Figs 9-14—[9—Vanadium exposure showed clear lumen in seminal vesicles of rat. 10—Normal secretion was noted in the lumen of seminal vesicles after conjoint treatment of T+Se. 11—Uterus of vanadium exposed rats showed degenerative changes in stroma. 12—Treatment with T+Se showed normal endometrium of uterus and uterine glands. 13—Ova of secondary follicle

showed high disruption after vanadium exposure. 14—Normal ova were clearly seen in the mature follicles with T+Se treatment] (Figs 9 &10: ×60; Figs 11 & 12: ×120; Figs 13 & 14: ×200).


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decreased fertility, embryolethality, fetotoxicity and teratogenicity in rats, mice and hamsters30. It gets accumulated in the testicles and reduces the motility of spermatozoa and other morphological distur-bances31-33. Fructose is secreted by the seminal vesicles, which is about 84% of the total fructose of the body34. In this study, it was decreased during toxicity, which may reduce the viability of spermatozoa due to improper nutrition.

Lipoic acid is an antioxidant, it can penetrate the cell membrane to exert therapeutic action by scavenge free radicals and chelate toxic heavy metals. It enhances the effects of glutathione35. Vitamin E is an antioxidant and also chelates heavy metals. Selenium is an important nutritional supplement, which works closely with vitamin E and supports critical antioxidant enzyme function. The therapeutic effec-tiveness (TEF) of tiron is approximately equal to 136, this chelator significantly increased urinary excretion of V, and the concentration of the metal was significantly reduced in kidney, liver and heart. Tiron was also reported to be an effective antidote for various metals viz. vanadium, beryllium and aluminium intoxication36-38. In the present study, treatment with tiron along with supplements LA, vit E and Se showed recovery pattern in various parameters. Efficacy of tiron to mobilize V to restore altered biochemical parameters could be attributed to the available binding sites and stability constant of the metal chelator complex. Tiron has four coordination numbers. One molecule of tiron may replace their hydrogen atoms and bind to V with its oxygen atom thereby forming a stable complex. This study has clearly shown that Tiron + Se is the more effective combination for vanadium intoxication. Se reduced the oxidative stress whereas Tiron remove metal. Therefore, further investigations are required before the possible use of this compound in clinical V toxicity.

Acknowledgement

The author (SS) is thankful to (UGC) and (ICMR) New Delhi for financial assistance.

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chelation therapy and vanadium: effect on reproductive

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