Effect of lindane on testicular antioxidant system and steroidogenic enzymes in adult rats

R. Sujatha, K.C. Chitra, C. Latchoumycandane, P.P. Mathur

School of Life Sciences, Pondicherry University, Pondicherry 605 014, India

Asian J Androl  2001 Jun; 3:  135-138

Keywords: lindane; rats; testes; antioxidants; steroids

Abstract

Aim: To find out the effect of lindane on testicular antioxidant system and test icular steroidogenesis in adult male rats. Methods: Adult male rats were orally administered with lindane at a dose of 5.0 mg/kg body weight per day for 30 days. Twenty-four hours after the last treatment the rats were killed using anesthetic ether. Testes, epididymis, seminal vesicles and ventral prostate were removed and weighed. A 10% testicular homogenate was prepared and centrifuged at 4.The supernatant was used for various biochemical estimations. Results: The body weight and the weights of testes, epididymis, seminal vesicles and ventral prostate were reduced in lindane-treated rats.  There was a significant decline in the activities of antioxidant enzymes superoxide dismutase (SOD), catalase and glutathione reductase while an increase in hydrogen peroxide (H₂O₂) generation was observed.  The specific activities of testicular steroidogenic enzymes 3-hydroxysteroid dehydrogenase and 17-hydroxysteroid dehydrogenase were decreased.  The levels of DNA, RNA and protein were also decreased in lindane-treated rats. Conclusion: Lindane induces oxidative stress and decreases antioxidant enzymes in adult male rats.

1 Introduction

The organochlorine insecticide lindane (-hexachlorocyclohexane) is widely used as a pesticide in many countries. In India it is widely used in agriculture and public health progammes.  Lindane possesses lipophilic character and enters into the food chain resulting in bioaccumulation in the body tissues, blood and breast milk of humans and wildlife^[1]. Lindane and other pesticides are released into the environment intentionally and exposure to such pesticides interacts with the mammalian endocrine system and may cause adverse effects on reproductive functions in wildlife and humans^[2].Lindane  has been previously shown to cause toxic effects on the testes of rats. In female rats lindane exerts antiestrogenic activity by disrupting estrous cycle with a reduction in the uterine weight^[3]. Lindane  has been reported to affect the development of mouse embryos in vitro in a dose-dependent manner^[4].

Lindane when given orally to mice during early, mid and late pregnancy has been reported to cause total absence of implantation sites, total resorption of fetuses, and low birth weight of pups with an increase in number of dead fetuses, respectively^[5]. The testes are highly susceptible to lindane as it crosses the blood-testis barrier and depresses spermatogenesis with a numeric reduction in spermatids and fragmentation of Sertoli cells^[6].  A few  toxicological studies have addressed the possible relationship between reproductive toxicity and exposure to chemicals that generate reactive oxygen species (ROS)^[7].

2 Materials and methods

2.1 Chemicals

Lindane (-hexachlorocyclohexane) was a gift from Jayakrishna Pesticides, Salem, Tamil Nadu, India.  Androsterone 3, 17 dione, p-nitrophenyl phosphate, p-nitrophenol and dehydroepiandrosterone  were purchased from Sigma Chemical Co. (St.Louis, Mo., USA).   All the chemicals used were of analytical grade obtained  from  local commercial  sources.

2.2 Animals and treatment

Wistar male rats (10-12 weeks of age) were obtained from the Central Animal Facility of the  Jawaharlal Institute of Postgraduate Medical Education and Research (JIPMER), Pondicherry, India. The rats were maintained under a well-regulated light and dark (12h-12h) schedule at 243 and were allowed free access to laboratory chow and tap water.  Lindane was dissolved in olive oil at a concentration of 5.0 mg/mL and the test group was given by oral intubation of lindane at a dosage of 5.0 mg/kg body weight/day for 30 days. The control animals received a similar volume of the vehicle alone. Twenty-four hours after the last treatment the rats were weighed and killed using anesthetic ether. Testes, epididymis, seminal vesicles and ventral prostate were removed,  cleaned of the adhering tissues and weighed. A 10% testicular  homogenate was prepared in normal saline using Polytron homogenizer and the homogenate was centrifuged at 800g for 30 min at 4. The supernatant was used for various biochemical assays.Protein was estimated by the method of Lowry^[17]. Estimations of superoxide dismutase^[18], catalase^[19], glutathione reductase^[20] and  hydrogen peroxide generation assay^[21] were done. The activities of steroidogenic enzymes 3-hydroxysteroid dehydrogenase and 17-hydroxysteroid dehydrogenase were estimated according to Bergmeyer^[22]. The extraction and determination of DNA and RNA were carried out following the technique of Schneider^[23].

2.3 Statistical analysis

3 Results

The body weights of lindane-treated rats did not show any significant changes during the course of the treatment. However, the body weights were significantly decreased at the end of the treatment as compared to the corresponding group of control animals (Figure 1). The weights of the testis, epididymis, seminal vesicles and ventral prostate were decreased significantly from those of the control group of animals (Table 1). 

Figure 1. Effect of lindane on the daily body weight changes of adult male rats for 30  days. The body weights of lindane-treated rats did not show any significant changes during the course of the treatment. However, the body weights were significantly decreased at the end of the treatment as compared to the corresponding group of control animals, ^bP<0.05.

Table 1.  Effect of lindane on the body and organ weights in male rats. n=6, means.^bP<0.05 vs the control group.

The specific activities of superoxide dismutase, catalase and glutathione reductase decreased while the levels of hydrogen peroxide was found to be elevated in treated rats when compared to the corresponding group of control animals. The specific activities of steroidogenic enzymes, 3-hydroxysteroid dehydrogenase and 17-hydroxysteroid dehydrogenase were decreased significantly (P<0.05). There was a significant decrease in the levels of testicular DNA, RNA and protein in treated group when compared to the control animals (Table 2).

Table 2.  Effect of lindane on biochemical parameters in testes of male rats. n=6, means. ^bP< 0.05 vs the control group.

¹mol pyrogallol auto-oxidized/min per mg protein at 32.
²nmol H₂O₂ consumed/min per mg protein at 32.
³nmol  NADPH oxidized/min per mg protein at 32.
⁴nmol H₂O₂ consumed/min per mg protein at 32.
⁵mg/g wet weight of testes.
⁶nmol  NAD converted to NADH/min per mg protein at 32.

4 Discussion

Lindane has been classified by World Health Organization (WHO Report) under the category of technical products as being moderately hazardous and it is still used as a pesticide in many countries. A dose of 5 mg of lindane/kg body weight has been considered by WHO as No Observed Effect Level (NOEL). But in the present study the  testis weights of lindane-treated rats were significantly decreased. The testis has been shown to be highly susceptible to lindane as it crosses blood-testis barrier and depresses spermatogenesis^[6]. The decrease in the testicular weight of lindane-treated rats may be due to reduced tubule size, spermatogenic arrest and  inhibition of steroid biosynthesis of Leydig cells^[24].

The weights of epididymis, seminal vesicles and ventral prostate in lindane-treated rats were decreased. Several studies have shown that the epididymis and accessory sex organs require a continuous androgenic stimulation for preservation of their normal structural and functional integrity^[25].  Thus the slight reduction in the weight of the epididymis and accessory sex organs in the treated rats may be due to lower bioavailability of androgens^[26].

The antioxidant system plays an effective role in protecting testes and other biological tissues below a critical threshold of reactive oxygen species thus preventing testicular dysfunction^[27].  Antioxidant enzymes constitute a mutually supportive team of defence against reactive oxygen species (ROS). In the present study the activities of antioxidant enzymes, superoxide dismutase, catalase and glutathione reductase were decreased in lindane-treated rats. The levels of hydrogen peroxide generation  were found to be elevated which serves as a marker of increased ROS. Thus in the present study an increase in hydrogen peroxide with a reduction in antioxidant enzymes indicates the oxidative stress induced by lindane. The decreased activities of steroidogenic enzymes 3-hydroxysteroid dehydrogenase and 17-hydroxysteroid dehydrogenase were indicative of the reduced testicularsteroidogenesis. Lindane has been shown to inhibit steroidogenesis by reducing steroidogenic acute regulatory protein (StAR) expression, an action that has contributed to the pathogenesis of lindane induced reproductive dysfunction^[28].The reduction of superoxide dismutase suggestis that it is involved in antioxidant defence and it has been shown to act as an alternate regulatory switch in testicular steroidogenesis^[29]. The cytochrome P450 enzymes of the steroidogenic pathway are known to produce free radicals. These free radicals are produced as a result of electron leakage due to the interaction of steroid products or other pseudosubstrates with the enzymes. The inability of the pseudosubstrate to be oxygenated promotes the release of reactive oxygen species^[30]. In lindane-treated rats the decrease in the testicular contents of DNA, RNA and protein were observed, which shows that reactive oxygen species can attack vital components of the cell-like nucleic acids and proteins.

Acknowledgements

References

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Correspondence to: Dr. P.P. Mathur, School of Life Sciences, Pondicherry University, Pondicherry 605 014, India.
Tel: +91-413-655 212   Fax: +91-413-655 212
E-mail: ppmathur@pu.pon.nic.in or  ppmathur@yahoo.com
Received 2000-11-17     Accepted 2001-04-03