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Chronic
effect of endosulfan on the testicular functions of rat
K.C.
Chitra, C. Latchoumycandane, P.P. Mathur
School
of Life Sciences, Pondicherry University, Pondicherry 605 014, India
Asian J Androl 1999 Dec; 1: 203-206
Keywords: pesticides; endosulfan; testis; steroidogenesis; rats; estrogens
Abstract
Aim: To find out the toxic effect of endosulfan on the testicular function of pubertal rats. Methods: Male rats of pubertal age were orally administered endosulfan at a dose of 1.0 mg/kg body weight for 30 days. Twenty-four hours after the last treatment, the rats were sacrificed and the testis, epididymis, seminal vesicles and ventral prostate were removed and weighed. A 10% testicular homogenate was prepared for biochemical estimations. Results: In endosulfan-treated rats, there were a reduction in the body weight and the weights of testis and accessory sex organs, a decrease in the testicular lactate and pyruvate activities, and in the testicular DNA and RNA concentrations, whereas the testicular protein concentration was slightly increased; the specific activity of testicular steroidogenic enzyme, 3-OH-steroid dehydrogenase and the ascorbic acid level were decreased, which were correlated with a decrease in steroidogenesis. The lysosomal enzyme acid phosphatase and brush-order enzyme alkaline phosphatase activities were also decreased in the testis of treated rats. Conclusion: In pubertal rats, endosulfan treatment inhibits the testicular functions.1 Introduction
There is growing concern that environmental chemicals, both natural and man-made, having estrogenic property may be causing a variety of reproductive disorders in wildlife and human populations. Most of the chemicals, which are used as pesticides are not highly selective, but are generally toxic to many non-target species, including man and other animals. Endosulfan, an insecticide of cyclodiene group is extensively used as an insecticide mainly in agriculture and in some countries in public health[1]. As a result of its wide spread use, it can be a potential environmental contaminant and may cause a public health hazard[2]. Endosulfan has been shown to cause embryonic death, teratogenesis, and inhibited embryonic growth[3]. Endosulfan undergoes oxidation to form a primary insecticidal metabolite, endosulfan sulfate, which is as toxic as endosulfan. The formation of endosulfan sulfate has been indicated in plants and in mice[4]. Endosulfan possesses estrogenic property and affects the differentiation and function of accessory sex organs in the males[4]. Exposure to estrogen or estrogenic chemicals like endosulfan during fetal or perinatal periods induced major pathological effects in the head of the epididymis both in the humans and experimental animals[5,6]. Endosulfan has been shown to reduce plasma FSH, LH, and testosterone in rats[7] and cause testicular damage[8]. The dose and duration of exposure to endosulfan are important determinants in its cellular and biochemical toxicity[7]. The biochemical alterations of the testis have been reported to be reversible after endosulfan withdrawal[7]. Oral administration of endosulfan in adult male rats decreased the sperm counts in the caudal epididymis and the intratesticular spermatid counts, associated with an elevation in the activities of specific testicular marker enzymes, as sorbitol dehydrogenase, lactic dehydrogenase, -glutamyl transpeptidase and glucose 6-phosphate dehydrogenase[7]. Endosulfan impaired the testicular functions through altering the activities of relevant enzymes[9]. Endosulfan also inhibited testicular androgen biosynthesis[10]. It is well known that the estrogenic action of endosulfan would injure male reproduction, whereas its direct toxicity on the male reproductive system has not been clearly shown. The present study was undertaken to evaluate the effect of environmental estrogenic compound, endosulfan, on the testicular function of pubertal rats.2 Materials and methods
2.1
Endosulfan
Endosulfan
(Thiodan, 6,7,8,9,10,10-hexachloro-1,5,5a,6,9,9a-hexahydro-6,9-methano-2,4,3-benzodioxathiepin-3-oxide)
was purchased from
SPIC India
Ltd. at
35% concentration.
2.2
Animal and treatment
Wistar male rats of pubertal age (45 days old) were obtained from the Central Animal Facility of the Jawaharlal Institute of Postgraduate Medical Education and Research (JIPMER), Pondicherry, India.
The
rats were divided at random into two groups of 6 animals each. They were
maintained under a well regulated light and dark (12 h:12 h) schedule
at 243, and were
allowed free access to laboratory chow and tap water. The test group was
given by oral intubation endosulfan dissolved in groundnut oil at a dose
of 1.0 mg/kg body weight for 30 days.
The control animals received a similar volume of the
vehicle. Twenty-four
hours after the last treatment, the rats were weighed and
sacrificed using ether anesthesia. The testis, epididymis, seminal vesicles and
ventral prostate were removed immediately, cleaned and weighed.
The testes were
homogenized using Remi Potter-Elvehjem homogenizer.
A 10 % homogenate was prepared in normal saline and the supernatant
used for biochemical assay. The extraction and determination of DNA and
RNA were carried out following the technique of Schneider[11].
The protein contents were estimated by the method of Lowry et al[12].
Estimations of ascorbic acid was performed according to Fisher[13],
lactate to Gutmann and Wahlefeld[14], pyruvate to Czoke and
Lampercht[15], 3-OH-steroid dehydrogenase to Bergmeyer[16],
and acid phosphatase and alkaline phosphatase to Torriani[17].
2.3
Data analysis
The data were presented as means. Statistical analysis was performed using Student's t test[18]. Significance of differences was set at P<0.05.
3 Results
The
body weight and the weights of the testis, epididymis, seminal vesicles,
and ventral prostate
were significantly lower (P<0.05) in the endosulfan-treated
rats than in the controls (Table 1).
Table
1. Effect of endosulfan
on the body and organ weights.
n=6. means. bP<0.05 vs the
control group.
|
|
Body
weight (g) |
Testis
weight (mg) |
Organ
weights (mg/100g body weight) |
||
|
Epididymis |
Seminal
vesicles |
Ventral
prostate |
|||
|
Control |
1319 |
95227 |
20910 |
58367 |
15350 |
|
Treated |
11812b |
78977b |
17744b |
307143b |
11518b |
From
Table 2 it can be seen that in the treated rats, the testicular contents
of DNA and RNA were
significantly decreased while the protein contents significantly increased
as compared with the controls; The activities of lactate, pyruvate, 3-OH-steroid
dehydrogenase, acid phosphatase and alkaline phosphatase were significantly
lower in the treated than in the control rats. The ascorbic acid level
was also lower in the treated animals.
Table
2. Effect of endosulfan
on testicular biochemistry.
means. n=6. bP<0.05 vs control
group.
|
Parameters |
Control |
Treated |
| Protein1 |
10.562.90 |
13.121.89b |
| DNA1 |
11.131.35 |
2.190.95b |
| RNA1 |
3.190.23 |
0.930.21b |
| Ascorbic
acid1 |
2.560.81 |
1.640.19b |
| Lactate2 |
45.087.97 |
32.649.97b |
| Pyruvate2 |
31.278.70 |
14.522.70b |
| 3-OH-steroid
dehydrogenase3 |
3.801.04 |
1.880.31b |
| Acid
phosphatase4 |
101.0725.2 |
79.2618.45b |
| Alkaline
phosphatase4 |
90.5938.42 |
55.6115.28b |
1mg/g
wet weight of testes
2nmol/mg protein
3nmol of NAD converted to NADH/min/mg protein
4mol p-nitrophenol liberated/35/30 min/100g protein
4 Discussion
Endosulfan
was classified by the WHO in the category of technical products that are
moderately hazardous. It has been shown that endosulfan has estrogenic
property[4] and male rats are more sensitive to the chronic
effect of endosulfan than female
rats[19]. In
the present experiments the body weights of endosulfan-treated rats decreased
significantly. The decrease in testicular weight of endosulfan-treated
rats may indicate impairment at testicular, pituitary, or hypothalamic
level. The epididymis, seminal vesicles and ventral prostate are all androgen-dependent
organs, relying on testosterone for their growth and function[20].
A reduction in their weights may reflect a decreased bioavailability and
production of androgens[21].
The
present study shows an increase in testicular protein in endosulfan-treated
rats, while the nucleic acids, DNA and RNA concentrations in the testis
are significantly decreased. The testicular fluid contains both stimulatory
factor as well as inhibitory factors, that selectively alter the protein
secretions[22]. Thus
the changes in protein, DNA and RNA suggest that there is a reduction
in the synthetic activity in testis.
Lactate and pyruvate play an important role in the metabolism of
testis[23]. FSH
has been reported to stimulate pyruvate production
in the rat Sertoli cells and this is a rate limiting factor for germ cell
activities[24]. The
decreased lactate and pyruvate activities may reflect the
reduced metabolic activities of the testis.
A
decrease in the activity of key steroidogenic enzyme, 3-OH-steroid dehydrogenase
reflects decreased steroidogenesis in the testis[25].
Ascorbic acid is
present in the spermatogenic chamber and Leydig cells[26].
Ascorbic acid in semen
has been shown to play an important role in preventing oxidative damage
to the spermatozoa[23].
A decrease in the concentration of ascorbic acid in the testis
may reflect a decrease in testicular steroidogenesis[27].
Acid phosphatase, an enzyme of a lysosomal origin is detectable
in all germinal cells, and its specific activity increases with the development
of spermatocytes[28].
Activities of free lysosomal enzymes have been shown to rise when
testicular steroidogenesis is increased[21].
A decrease in the acid phosphatase in free state would thus reflect
decreased testicular steroidogenesis in the treated-rats and this may
be correlated with the reduced secretion of gonadotrophins.
A decrease in the alkaline phosphatase activity in endosulfan-treated
rats indicated that endosulfan treatment produced a state of decreased
steroidogenesis where the inter- and intracellular transport was reduced
as the metabolic reactions to channalize the necessary inputs for steroidogenesis
slowed down[29].
5
Acknowledgments
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Correspondence to Dr. P. P. Mathur
E-mail: pmathur@yahoo.com
Tel:
+91-413-655 212 Fax: +91-413-655 211
Received
1999-09-15 Accepted 1999-11-18
