Effect of mercuric chloride on membrane-bound enzymes in rat testis

Asian J Androl 2002 Dec; 4: 309-311           

Keywords:mercuric chloride; testis; alkaline phosphatase; 5' nucleotidase; g-glutamyl transferase; adenosine triphosphatase

Abstract

Aim: To study the effect of mercuric chloride on the membrane-bound enzymes. Methods: The effect of mercuric chloride at two different doses, 1 mg/kg (low dose) and 2 mg/kg (high dose), orally for 30 days, was observed on the membrane-bound enzymes in the testis of adult albino rats. Results: Mercuric chloride significantly decreased the body weight and testis weight in the high dose group (P< 0.05), but not in the low dose group. The activities of 5' nucleotidase and adenosine triphosphatases were markedly decreased (P< 0.01) in the testis of both groups. Alkaline phosphatase and g-glutamyl transferase activities were significantly increased (P< 0.01) in both groups. However, the effect was more pronounced in the high than in the low dose groups. Conclusion: The dose dependent effect of mercuric chloride on these enzymes may affect the membrane characteristics and thereby the fertility of the animal.

Mercuric chloride has been used in agriculture as a fungicide, in medicine as topical antiseptic and disinfectant and in chemistry as an intermediate in the production of other mercury compounds. It is evident that mercury released into the environment affects the reproductive system of several animals. Mercury elicited direct toxic action on steroid producing cells in the adrenal gland and testis [1]. Mercuric chloride treatment caused the degeneration of Leydig cells and diminished the activity of steroidogenic enzymes [2] and also resulted in the intracellular accumulation of mercury in the interstitial and Sertoli cells of the seminiferous tubules [3].

Administration of mercury to fowl resulted in hypospermia and the occurrence of abnormal spermatozoa [4]. Enhanced lipid peroxidation was observed in the testis of mercuric chloride treated rats [5]. Previous study in our laboratory has shown the adverse effect of mercuric chloride on testis, spermatozoa and brain [6-9]. However, the exact cellular mechanism by which mercury acts as a reproductive toxicant is poorly document-ed.

The plasma membrane receives and transduces growth promoting signals which trigger a cascade of physiological and biochemical events in the cell [10]. Considering the major role of plasma membrane in cellular functions and the plasma membrane may be the target of metal toxicity, the present study was conducted to evaluate the effect of mercuric chloride on testicular plasma membrane enzymes.

2 Materials and methods

2.1 Animals and grouping

Old male Wistar rats (90 days, 185 g~200 g), purchased from the animal center of Jawaharlal Nehru Institute of Post Graduate Medical Education and Research, Pondicherry, were used in the present investigation. They were housed in plastic cages in a room with controlled temperature (251), humidity (50 %5 %) and lighting (12 h light and 12 h dark). The rats were fed with standard commercial dry food pellets (Gold Mohur, India) and tap water ad libitum. After 10 days adaptation period the rats were divided into three groups of 15 animals each.

Group I: Control - gavaged 0.5 mL of distilled water (vehicle) per day, orally for 30 days.

Group II: Low dose - gavaged mercuric chloride 1 mg.kg^-1.day^-1 (in 0.5 ml distilled water), orally for 30 days.

Group III: High dose - gavaged mercuric chloride 2 mg.kg^-1.day^-1 (in 0.5 ml distilled water) orally for 30 days.

2.2 Treatment and observation

Before drug administration the food intake was measured daily for 15 days and the body weight was recorded. In the treatment period, the food consumed every 5 days was measured; the body weight was measured on every 5th day and the percent changes were calculated.

The animals were treated between 8 am and 9 am. After the 30 days experimental period, all the rats were sacrificed by decapitation. The testis (right) was dissected and accurately weighed. The testicular plasma membrane was isolated by the method Dalterio et al reported [11]. The activities of ATPases EC 3.6.1.3 [12], alkaline phosphatase EC 3.1.3.1 [13], 5' nucleotidase EC 3.1.3.5 [14] and g-glutamyl transferase EC 2.3.22 [15] in the plasma membrane were estimated spectrophotometrically. Enzyme activities are expressed in terms of specific activity as standard units/mg protein. Protein content was estimated by the method of Lowry et al [16].

2.3 Statistical analysis

Results are expressed as mean SEM. The data were statistically analyzed by the Student's t-test described by Fisher. P< 0.05 was assumed significant.

3 Resilts

Mercuric chloride treatment decreased the body weight and testis weight (P< 0.05) only in the high dose group, but not in the low dose group (Table 1). The activities of alkaline phosphatase and g-glutamyl transferase were increased (P<0.01) in the low dose treated group and further increased (P<0.001) in the high dose treated group. However, the activities of 5' nucleotidase, Ca²⁺, Mg²⁺ and Na⁺K⁺- ATPases were decreased in both the low dose (P< 0.05) and the high dose groups (P<0.001) (Table 2).

Table 1. Effect of mercuric chloride on body weight and testis weight in rats.^bP< 0.05, compared with controls.

Table 2. Effect of mercuric chloride on membrane-bound enzymes in rat testis. bP<0.05, cP<0.01, compared with controls.

4 Discussion

The decrease in the body weight observed due to mercuric chloride administration may be due to the reduced food and water intake. This is in accordance with the report of National Toxicology Program Working Group [17] that mercuric chloride treatment to rats resulted in reduced food and water intake leading to weight loss. The decreased testicular weight observed shows the degenerative effect of mercuric chloride.

Most toxicants interact with the intracellular target molecules altering the cellular metabolism and function, which eventually leads to cell membrane damage and cell lysis. Metal-induced plasma membrane damage occurs directly through interaction with the membrane components as the ion dependent ATPases and ion channels and indirectly as a consequence of overt cytosolic damage. Metals generally inhibit the function of ion dependent ATPases leading to disturbances in the ion homeostasis. Disturbances in the ion homeostasis results in impaired signal transduction, altered cellular metabolism, changes in cell membrane permeability and integrity, and disturbances of vital function.

Alkaline phosphatase is involved in the synthesis of nuclear proteins, nucleic acids and phospholipids as well as in the cleavage of phosphate esters and in mobilizing carbohydrates and lipid metabolites to be utilized either within the cells of the accessory sex structure or by the spermatozoa in the seminal fluid. 5' nucleotidase is an established plasma membrane marker in many mammalian cells where it exists as an ectoenzyme [18].

g-glutamyl transferase is another membrane-bound enzyme, which catalyzes the transfer of g-glutamyl group between peptides and amino acids [19]. g-glutamyl transferase is considered a marker enzyme of Sertoli cell function of testis and its activity parallels the pattern of Sertoli cell maturation and replication [20]. The significant increase in the activity of g-glutamyl transferase in the testis indicates an impaired function of Sertoli cells by the treatment of mercuric chloride. The disturbed ion homeostasis due to the inhibition of ATPases and 5' nucleotidase in the testis of rats treated with mercuric chloride may eventually lead to testicular cell death and atrophy. It is also possible that decreased ATPase activity may lead to decreased ATP breakdown and thereby the availability of free energy is reduced. Several metabolic processes may also be affected due to this change.

It is evident that mercuric chloride decreased the activities of ATPases and 5' nucleotidase while increasing the activities of alkaline phosphatase and g-glutamyl transferase. From the present investigation it is clear that mercuric chloride has a definite, dose dependent and differential influence over plasma membrane enzymes in the testis of rat. This influence may bring about alterations in the physico-chemical properties of testicular membranes. Eventually this may have an impact on testicular functions and fertility of the animal.

References  

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Correspondence to: Dr. V.Ramalingam, Department of Zoology, Bharathidasan Govt. College for Women, Pondicherry - 605 003, India.

E-mail: ramalingamv@yahoo.com

*5 Lancelot Court # 4, Salem, NH 03079, USA

Received 2001-09-29      Accepted 2002-10-29