:: Asian Journal of Andrology ::

Effect of lindane on antioxidant enzymes in epididymis and epididymal sperm of adult rats

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

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

Asian J Androl 2001 Sep; 3: 205-208

Keywords: lindane; -hexachlorocyclohexane; epididymis; antioxidants; reactive oxygen species

Abstract

Aim: To find out the changes induced by lindane on the antioxidant enzymes in epididymis and epididymal sperm of adult rats. Methods: Adult male rats were orally administered lindane at a dose of 5.0 mg/kg body weight per day for 30 days. At the end of the treatment, the rats were sacrificed. The epididymis was removed and weighed and sperm were collected for sperm count, motility and biochemical studies. A 1% homogenate of epididymis was prepared and used for biochemical estimations. Results: In lindane-treated rats, there were significant reductions in the epididymal weight, epididymal sperm count and motility compared with the controls. Significant decreases in the superoxide dismutase (SOD), catalase, glutathione reductase and glutathione peroxidase activities and significant increases in the H₂O₂ generation and lipid peroxidation were also observed in the epididymis and epididymal sperm of lindane-treated rats. Conclusion: Lindane decreases the levels of antioxidant enzymes in the epididymis and epididymal sperm of adult rats thereby inducing oxidative stress.

1 Introduction

Lindane, the -isomer of 1,2,3,4,5,6-hexachlorocyclohexane (-HCH), is one of the most widely used organochlorine insecticide in India. There is much concern that exposure to estrogen- or estrogen-like chemicals induce major pathological effects in the epididymis, in men and experimental animals. Lindane possesses estrogenic^[1]as well as antiestrogenic^[2] properties and has been shown to affect the differentiation and function of epididymis in rats^[3]. Many environmental contaminants have been reported to disturb the pro-oxidant/antioxidant balance of the cells by generating oxygen free radicals and reactive oxygen species (ROS) thereby inducing oxidative stress^[4,5]. ROS have been shown to damage almost all the macromolecules of the cell including membrane bound polyunsaturated fatty acids (PUFA), causing impairment of cellular functions^[6]. The epididymisis highly rich in PUFA and therefore susceptible to the oxidative stress. Exposure to environmental contaminant lindane has been shown to enhance oxidative stress in liver and testis^[7,8]. The present study was undertaken to evaluate the effects of lindane on the antioxidant enzymes of epididymis and epididymal sperm in adult rats.

2 Materials and methods

2.1 Chemicals

Lindane (99%) was a gift from Jayakrishna Pesticides, Salem, Tamil Nadu, India. All other chemicals used for various assays were of analytical grade and obtained from local commercial sources.

2.2 Animals and treatment

Adult Wistar male rats (10-12 weeks) were obtained from the Central Animal House of the Jawaharlal Institute of Postgraduate Medical Education and Research (JIPMER), Pondicherry, India. Animals were maintained in plastic cages under a well-regulated light and dark (12 h:12 h) schedule at (243) . Standard commercial laboratory chow and tap water were given ad libitum. Lindane (5.0 mg/mL, in olive oil) was given orally at a dosage of 5.0 mg/kg body weight per day for 30 d. The control animals received a similar volume of the vehicle. Body weights were registered on every alternate day until the end of the experiment. The animals were killed by ether 24 h after the last treatment. Epididymis was removed, cleared off the adhering tissues and weighed. The epididymal sperm counts and motility were done immediately and the remaining sperm were collected and used for biochemical assays.

2.3 Collection of epididymal sperm

The epididymal sperm were collected by cutting epididymis into small pieces and flushing the sperm in normal saline. The sperm collected was centrifuged at 225g for 10 min. The pellet was resuspended in 2.0 mL of normal saline. An aliquot of sperm suspension was homogenized for a few seconds, centrifuged at 800g for 10 min and used for biochemical assays.

2.4 Epididymal sperm count and sperm motility

Epididymal sperm counts and evaluation of the motility of epididymal sperm were done by the method of Linder et al^[9]. The epididymal sperm was obtained as described above and incubated at 32 , which is the optimum temperature of rat epididymal sperm. The epididymal fluid was then diluted to a volume of 5 mL of pre-warmed (32 ) normal saline. An aliquot of this solution was placed in Neubauer hemocytometer and motile sperm were counted by using microscope. Non-motile sperm numbers were first determined, followed by counting of total sperm. Sperm motility was expressed as a percent of motile sperm of the total sperm counted.

2.5 Biochemical assays

A 1% homogenate of epididymis was prepared, centrifuged at 800g for 10 min and the supernatant used for biochemical assays. In the epididymis as well as in epididymal sperm, the biochemical assays of catalase^[10], superoxide dismutase^[11], glutathione reductase^[12], glutathione peroxidase^[13], hydrogen peroxide generation assay^[14] and lipid peroxidation^[15] were estimated. Protein was estimated by the method of Lowry et al^[16], and DNA by the method of Burton^[17].

2.6 Statistical analysis

The data were presented as meanS.D. Statistical analysis was performed using Student's t-test. Significance of differences was set at P<0.05.

3 Results

The weight of the epididymis was significantly decreased (P<0.05) in the lindane-treated rats to (32824) mg from the control value of (48816) mg when compared to the corresponding group of control animals. The weight of epididymis when expressed relative to the body weight was also found to be significantly (P<0.05) decreased to (25620) mg/100g body weight from the control value of (28944) mg/100g body weight. In the lindane-treated rats, the epididymal sperm counts were significantly (P<0.05) decreased to (3.300.21)10⁸ from the control value of (4.090.13) 10⁸ per epididymis. The epididymal sperm motility were significantly (P<0.05) decreased to 26.33 %10.67 % from the control value of 81.66 %13.43 %.

The specific activities of catalase, superoxide dismutase, glutathione reductase, glutathione peroxidase were found to be decreased significantly while the level of hydrogen peroxide generation and lipid peroxidation were increased significantly (P<0.05) in the epididymis of lindane-treated rats when compared to the control animals (Table 1). The biochemical estimations were done in caput, corpus and cauda epididymis, where cauda epididymis showed more significant changes than the other two regions (data not shown). In the epididymal sperm of lindane-treated rats the specific activities of catalase, superoxide dismutase, glutathione reductase, glutathione peroxidase were found to be decreased significantly whereas the level of hydrogen peroxide generation and lipid peroxidation were increased significantly when compared to the corresponding group of control animals (Table 2). A definite correlation (r=0.8; n=12) was observed between sperm counts and DNA content of epididymal sperm so that the DNA content was routinely used as an indicator of sperm counts. The results were thus expressed both in terms of protein and DNA.

Table 1. Effect of lindane on antioxidant enzymes in cauda epididymis of adult rats.

Parameters	Control	Treated
Catalase^a	2.770.13	1.170.25^*
Superoxide dismutase^b	15.01.52	7.611.77^*
Glutathione reductase^c	28.12.71	19.70.86^*
Glutathione peroxidase^c	40.712.2	29.87.01^*
H₂O₂ generation assay^d	0.380.02	1.100.08^*
Lipid peroxidation^e	3.750.20	5.060.50*

^*P<0.05 vs the control group
^a mol H₂O₂ consumed/ min/ mg protein at 32
^bnmol pyrogallol oxidized/ min/ mg protein at 32
^cnmol NADPH oxidized/ min/ mg protein at 32
^dnmol H₂O₂ generated/ min/ mg protein at 32
^emol malondialdehyde produced/ min/ mg protein

Table 2. Effect of lindane on antioxidant enzymes in epididymal sperm of adult rat (n=6).

Parameters	Control	Treated
Catalase^a
(mg protein)	3.920.18	2.820.27^*
(mg DNA)	1.810.12	1.400.16^*
Superoxide dismutase^b
(mg protein)	26.542.38	6.671.12^*
(mg DNA)	20.302.82	12.51.49^*
Glutathione reductase^c
(mg protein)	28.452.73	24.131.48^*
(mg DNA)	12.200.69	10.000.56^*
Glutathione peroxidase^c
(mg protein)	30.263.12	22.482.98^*
(mg DNA)	26.303.35	19.901.12^*
H₂O₂ generation assay^d
(mg protein)	1.170.28	1.710.22^*
(mg DNA)	0.530.12	0.780.09^*
Lipid peroxidation^e
(mg protein)	5.280.23	5.610.44^*
(mg DNA)	4.220.37	5.740.39^*

^*P<0.05 vs the control group
^amol H₂O₂ consumed/ min at 32
^bnmol pyrogallol oxidized/ min at 32
^cnmol NADPH oxidized/ min at 32
^dnmol H₂O₂ generated/ min at 32
^emol malondialdehyde produced/ min

4 Discussion

Lindane is an environmental contaminant classified by World Health Organization as moderately hazardous^[18]. There is much concern that exposure to such environmental contaminants causes decreased sperm counts, impairment of sperm motility, reduced fertilization ability, producing abnormal sperm in men and wildlife^[19]. Our previous studies on one of the environmental contaminants endosulfan have shown to cause impairment of testicular functions and affect the androgenicity in rats^[20]. A dose selected in the present study (5.0 mg of lindane/ kg body weight) has been considered as No Observed Effect Level (NOEL)^[18]. Lindane treatment has shown a significant decrease in the body weight and the weights of testis, ventral prostate and seminal vesicles^[8]. In the present studies the epididymal weight of lindane-treated rats were decreased significantly and this may be due to the decreased bioavailability and production of androgens^[20].

Pro-oxidant and antioxidant balance is vital for normal biological functioning of the cells. If any of the complex components such as environmental contaminants affecting this balance can provoke excessive production of ROS that is effectively scavenged by endogenous antioxidant defence system^[4]. In spermatozoa, several antioxidant systems as glutathione peroxidase^[21], superoxide dismutase^[22] and catalase^[23] are known to operate. Cytoplasm of spermatozoa is extremely limited in volume and localization, so the polyunsaturated fatty acids that bound in the sperm plasma membrane are very susceptible to ROS attack^[24].

Antioxidant enzymes constitute a mutually supportive team of defence against ROS. In the present study the specific activities of catalase, superoxide dismutase, glutathione reductase and glutathione peroxidase was found to be decreased while an increase in the hydrogen peroxide generation and lipid peroxidation were observed in epididymis and epididymal sperm of the lindane-treated rats. Thus in the present study the increase in lipid peroxidation with a reduction in antioxidant enzymes indicates that lindane induces oxidative stress in epididymis and epididymal sperm. The significant increase in the lipid peroxidation of epididymal sperm in lindane-treated rats has also been associated with the decreased sperm motility. The generation of ROS has been correlated with pathological sperm categories as oligospermia termed as defective sperms with reduced sperm motility, fertility and sperm-oocyte fusion^[25]. In the present study, the epididymal sperm counts and sperm motility were decreased in lindane-treated rats as compared to the control animals indicating that epididymis was undergoing oxidative stress.

In conclusion, the present studies reflect that lindane induces oxidative stress in rat epididymis and epididymal sperm by increasing ROS and decreasing the levels of antioxidant enzymes with a possible reduction in epididymal sperm counts and epididymal sperm motility.

Acknowledgements

The authors thank the staff of Bioinformatics Centre, Pondicherry University, Pondicherry for providing various facilities. KCC acknowledges Lady Tata Memorial Trust, Mumbai, India for the Junior Scholarship. CL acknowledges Indian Council of Medical Research, New Delhi, India for Senior Research Fellowship. PPM acknowledges the receipt of financial support from Population Council, New York, USA (Grant Nos. B99.047P-9/ ICMC and B99.048 R/ ICMC).

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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: +91-413-655 211
E-mail: ppmathur@pu.pon.nic.in; ppmathur@yahoo.com
Received 2001-084-10 Accepted 2001-08-27