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Effect
of methoxychlor on antioxidant system of goat epididymal sperm in vitro
Bindu
Gangadharan, M. Arul Murugan, P.P. Mathur
School
of Life Sciences, Pondicherry University, Pondicherry 605 014, India
Asian J Androl 2001 Dec; 3: 285-288
Keywords:
methoxychlor;
vitamin C; epididymis; spermatozoa; antioxidants; reactive oxygen species;
goats
Abstract
Aim:
1
Introduction
There
has been an increasing concern regarding the releasing of some natural
and synthetic chemicals into the environment causing disruption of normal
sexual development, differentiation and function in animals and humans[1].
Methoxychlor, a DDT substitute chlorinated hydrocarbon pesticide, is a
labile and readily degradable compound widely used in agriculture, livestock
industry, home and gardens[2]. Although methoxychlor does not
persist long in the environment, it may enter the body of grazing animals
and humans through the food chain. Continuous exposure to methoxychlor
results in reproductive abnormalities in amphibian[3], mice[4],
rats and other animals[5]. Methoxychlor possesses estrogenic
and antiandrogenic properties in vivo and in vitro[6].
Pesticides with such properties have been shown to cause overproduction
of reactive oxygen species(ROS) in both intra and extracellular spaces,
resulting in a decline of sperm count and infertility in wildlife and
humans[7].
Many environmental contaminants have been reported to disturb the pro-oxidant/antioxidant balance of the cells thereby inducing oxidative stress[8]. ROS has been shown to damage macromolecules, including membrane bound polyunsaturated fatty acids (PUFA), causing impairment of cellular function[9]. Spermatozoa are rich in PUFA and, therefore, could be highly susceptible to oxidative stress. Several investigators have reported that vitamin C, a powerful water-soluble antioxidant, protects against oxidative damage caused by free radicals[10]. Previous studies in our laboratory have shown that lindane, an environmental contaminant, alters the antioxidant system in testis[11] and epididymis and epididymal sperm[12] in rats. The present study was undertaken to evaluate if methoxychlor induces oxidative stress in the epididymal sperm of goats and the effect could be reversed by vitamin C.
2 Materials and methods
2.1
Chemicals
Methoxychlor 1,1,1-trichloro-2,2-bis (4-methoxyphenyl) ethane was a gift from Dr. Ute Tiemann, Research Institute for the Biology of Farm Animals, Wilhelm Stahl Allee 2, 18196 Dummerstorf, Germany. All other chemicals were of analytical grade and purchased from local commercial sources.
2.2 Sperm Collection
Spermatozoa were obtained from goat epididymis as described by Roy et al[13]. Briefly, epididymis was cut into 4-5 pieces with a sharp razor blade and dispersed in a modified Ringer's phosphate solution (RPS): (NaCl 119 mM, KCl 5 mM, MgSO4 1.2 mM, glucose 10 mM, potassium phosphate 16.3 mM, penicillin 50 units/mL; pH 6.9) with gentle stirring. The sperm obtained was washed several times in RPS and centrifuged at 225g for 10 min. An aliquot of the sperm suspension were homogenised for ten seconds in cold RPS medium using glass teflon homogenizer of Potter-Elvehjem type. An aliquot was centrifuged at 800g for 20 min at 4 . The supernatant was used for biochemical assays.
2.3 Sperm incubation
Sperm samples dispersed in RPS were incubated with methoxychlor (1 mol/M, 10 mol/M and 100 mol/M) and methoxychlor+vitamin C (100 mol/M each) for 3 h at 32.
2.4 Sperm motility and viability
After incubation, an aliquot of sperm suspension (5 L) was placed in the Neubauer haemocytometer. The percentage of motile sperm was counted under light microscope at 200 magnification. The viability of epididymal sperm was determined by eosin staining and counting under light microscope at 200 magnification. The data were expressed in percentage of total sperm.
2.5 Biochemical assays
After 3 hours of incubation, the biochemical assays of superoxide dismutase[14], glutathione reductase[15], glutathione peroxidase[16] and lipid peroxidation[17] were performed. DNA was estimated by the method of Burton[18].
2.6 Statistical analysis
The data were presented as meanSD, if applicable. Statistical analysis was performed using Student's t-test. Significance of differences was set at P<0.05.
3
Results
In methoxychlor-incubated sperm and in the sperm co-incubated with methoxychlor and vitamin C, the epididymal sperm motility and viability showed no significant changes as compared to the corresponding control sperm samples (Figure 1). The specific activities of superoxide dismutase, glutathione reductase and glutathione peroxidase were found to be decreased in methoxychlor-incubated sperm samples while the level of lipid peroxidation was increased in a dose-dependent manner when compared to the corresponding controls (Figure 2). When sperm samples were co-incubated with methoxychlor and vitamin C the specific activity of antioxidant enzymes superoxide dismutase, glutathione reductase, glutathione peroxidase and the level of lipid peroxidation remained unchanged (Figure 3).
Figure 1. Effect of incubation of sperm with methoxychlor and co\|incubation of sperm with methoxychlor and vitamin C on epididymal sperm motility and viability of goat sperm. The values are expressed as meanSD. The data were expressed in percentage of total sperm.
Figure 2. Effect of incubation of sperm with methoxychlor on antioxidant system in goat epididymal sperm. The values are expressed as meanSD. bP<0.05 vs control. The unit of superoxide dismutase is expressed as nmol pyrogallol oxidized/min/109 spermatozoa at 32. The unit of glutathione reductase and glutathione peroxidase is expressed as nmol NADPH oxidized/min/109 spermatozoa at 32. The unit of lipid peroxidation is expressed as mol malondialdehyde produced/15 min/109 spermatozoa.
Figure 3. Effect of co-incubation of sperm with methoxychlor and vitamin C on antioxidant system in goat epididymal sperm. The values are expressed as meanSD. The unit of superoxide dismutase is expressed as nmol pyrogallol oxidized/min/109 spermatozoa at 32. The unit of glutathione reductase and glutathione peroxidase is expressed as nmol NADPH oxidized/min/109 spermatozoa at 32. The unit of lipid peroxidation is expressed as mol malondialdehyde produced/15 min/109 spermatozoa.
4
Discussion
Methoxychlor
has been considered as a pro-estrogen metabolized into mono- and bis-hydroxymetabolites
which possess higher estrogenic activity than the parental compound[19].
The major metabolite of methoxychlor, HPTE (2,2-bis ( p-hydroxyphenyl
)-1,1,1-trichlo-roethane), has been shown to bind both the estrogen and
androgen receptors[20], causing estrogenic effect in the males[21].
Methoxychlor at a dose of 20 g/kg has been considered the No Observed
Adverse Effect Level (NOAEL) for humans[22]. The dosages selected
in the present study (1, 10 and 100
mol/L) were below the NOAEL and these doses did not significantly
decrease the sperm motility and viability when compared to the controls.
A few toxicological studies have reported that exposure to environmental contaminants generates ROS and causes adverse effects on male reproduction[23].
In
spermatozoa, several antioxidant defence systems, namely, glutathione
peroxidase[24], superoxide dismutase[25] and catalase[26]
are known to operate. Superoxide dismutase generally dismutases the superoxide
anion radical into hydrogen peroxide[27]. The hydrogen peroxide
produced during the reactions is removed by catalase or glutathione peroxidase/reductase
system[24]. It has been reported that increase in ROS can cause
the destruction of all cellular structures including membrane lipid[23].
Hence in the present study lipid peroxidation is used as an index of oxidative
stress. Malondialdehyde, a lipid peroxidation product generated in tissues
by free radical injury is measured by thiobarbituric acid reactivity and
has been considered as sensitive index of free radical generation[23].
The present study indicated that graded doses of methoxychlor induced oxidative stress thereby decreasing the activities of antioxidant enzymes in goat epididymal sperm. In conclusion, induction of oxidative stress by methoxychlor could be reversed by the antioxidant, vitamin C, in the epididymal sperm of goats.
Acknowledgements
The authors thank Dr. Ute Tiemann for the generous gift of methoxychlor and the staff of Bioinformatics Center, Pondicherry University, Pondicherry for providing various facilities. The authors also acknowledge the receipt of financial support from the Population Council, New York, USA (Grant Nos. B 99.047P-9/ ICMC and B99.048R/ 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@hotmail.com
Received 2001-10-16 Accepted 2001-11-26
