Endosulfan induced oxidative stress and biochemical changes in testes of mice

Abstract

Endosulfan is potent toxicogen and carcinogen. It is widely used in agriculture and detected in soil, surface water, and underground water, fruits, vegetables and milk. A number of studies have been performed to investigate the effect of endosulfan on reproductive system. Exposure to endosulfan leads to degeneration of spermatogenesis, lower testosterone level and induce oxidative stress. Swisss albino male mice were grouped as control and endosulfan treated for 4, 8, 12 weeks at animal house of S S Hospital and Research Institute, Patna. Sperm count was performed by routine process, testosterone level was determined by ELISA kit method, and lipid peroxidation was done by measuring MDA level in blood serum. Decrease in testicular weight, sperm count, testosterone level and increase in MDA level were observed when compared with control upon administration of endosulfan at the rate of 3 mg/kg/body weight with significance of (p-value<0.0001). The effect of endosufan on reproductive system is deleterious and insidious. Endosulfan has been used in agriculture widely and enters human body system through food chain to cause toxicity. Hence more studies and investigations are warranted in this field the mechanism by which endosulfan impairs the reproductive system.

Full text

Anshu et al. European Journal of Pharmaceutical and Medical Research
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600
ENDOSULFAN INDUCED OXIDATIVE STRESS AND BIOCHEMICAL CHANGES IN
TESTES OF MICE
A. Nath1, Aseem Kumar Anshu1*, Priyanka1, Chandan Kumar Singh1, Sachidananda Behera1, J. K. Singh2
1Research Insitute, S S Hospital and Research Institute, Patna.
2Department of Oncology, S S Hospital and Research Institute, Patna.
Article Received on 19/07/2016 Article Revised on 10/08/2016 Article Accepted on 31/08/2016
INTRODUCTION
Numerous new chemicals have been introduced in recent
years and are still being evaluated for their potentiality to
cause toxicity. A large number of chemicals which has
been used and studied extensively are currently re-
evaluated for their toxic effects on reproductive
system[1]. India is an agro-based country and unrestrained
used of pesticide is inevitable. One of the broad spectrum
insecticide, Endosulfan (6,7,8,9,10,10 hexachloro-1,5,5a,
6,9,9a-hexahydro-6,9-methano-2,4,3-benzodioxathiepin-
3-oxide) which is used extensively in agriculture as
pesticide to protect cotton, vegetables and fruit crops.
Endosulfan and its metabolites have been detected in
soil, surface water, and underground water[2], milk[3, 4],
fruits and vegetables[5,6]. Endosulfan enters in our body
through various routs like ingestion, inhalation, or skin
contact and has toxic effect on the body depending upon
routs of administration, species, vehicle, and sex[7].
Endosulfan, being an endocrine disruptor, has been
observed to induce alteration in the process of
spermatogenesis in young and adult rats[8,9]. Decreased
sperm count, intratesticular sperrmatids number, altered
sperm morphology, and activity of testicular marker
enzymes. Several reports demonstrate the effect of
different compounds including endosulfan suppressing
development of prenatal gonads and spermatogenesis in
male offspring[10]. A detailed study revealed that 2.5, 5,
and 10 mg/kg/body weight of endosulfan for 10 weeks
reduce the intratesticular sperrmids count, sperm
abnormalities, and changes in activities of marker
enzymes in testes like lactate dehydrogenase, sorbitol
dehydrogenase, ƴ-glutamyl transpeptidase, and glucose-
6-phosphate dehydrogenase which corroborate the
detrimental effect of endosulfan on spermatogenesis [11].
The human population has been exposed to the harmful
compounds like endosulfan. Our aim of the study is to
Demonstate the effect of endosulfan on the reproductive
system by testing on mice models. In this article, we
report the effect of endosulfan on male reproductive
system of mice.
MATERIALS AND METHODS
Animals: Swiss albino mice (Mus musculus) were reared
in the animal house of S.S. Hospital and Research
Institute, Patna. The male mice selected for the study
were 12 weeks old and their weight was measured as
30±2 grams. Mice were kept in the polypropylene cages
with paddy husk at room temperature 28±2ºC and
humidity 50±5% in a controlled light (12 hrs light and 12
hrs dark). Animals were maintained in ideal conditions
SJIF Impact Factor 3.628
Research Article
ISSN 3294-3211
EJPMR
EUROPEAN JOURNAL OF PHARMACEUTICAL
AND MEDICAL RESEARCH
www.ejpmr.com
ejpmr, 2016,3(9), 600-603
*Correspondence for Author: Aseem Kumar Anshu
Research Insitute, S S Hospital and Research Institute, Patna.
ABSTRACT
Endosulfan is potent toxicogen and carcinogen. It is widely used in agriculture and detected in soil, surface water,
and underground water, fruits, vegetables and milk. A number of studies have been performed to investigate the
effect of endosulfan on reproductive system. Exposure to endosulfan leads to degeneration of spermatogenesis,
lower testosterone level and induce oxidative stress. Swisss albino male mice were grouped as control and
endosulfan treated for 4, 8, 12 weeks at animal house of S S Hospital and Research Institute, Patna. Sperm count
was performed by routine process, testosterone level was determined by ELISA kit method, and lipid peroxidation
was done by measuring MDA level in blood serum. Decrease in testicular weight, sperm count, testosterone level
and increase in MDA level were observed when compared with control upon administration of endosulfan at the
rate of 3 mg/kg/body weight with significance of (p-value<0.0001). The effect of endosufan on reproductive
system is deleterious and insidious. Endosulfan has been used in agriculture widely and enters human body system
through food chain to cause toxicity. Hence more studies and investigations are warranted in this field the
mechanism by which endosulfan impairs the reproductive system.
KEYWORDS: Endosulfan, toxicogen, carcinogen.

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as per animal ethical guidelines of the CPCSEA,
(CPCSEA Regd. No. 1840/PO/ReBi/S/5/CPCSEA) Gov.
of India and Institutional Animal Ethics Committee
(IAEC).
Study Groups and Sampling: 6 male mice of control
group were given distilled water as drinking water and
‘treated group’ (n=24) were administered 3mg/kg body
weight of endosulfan for 4 weeks, 8 weeks and 12
weeks. Blood samples were collected from the sacrificed
mice and centrifuged to obtain serum samples for
estimation of Lipid Peroxidation and Testosteron level.
Testes from all the animals were removed and washed
three times in isotonic saline solution (0.85 v/w%) and
fixed in 10% formalin for histological study.
Body and Organ weight: Body weight of each group of
male mice was measured before and after the
administration of endosulfan. Each group of male mice
were sacrificed after 4 weeks, 8 weeks and 12 weeks and
testes were cut out and weighed.
Sperm Count: The epididymis was removed and
washed in isotonic saline. The semen was collected by
puncturing epididymis at several places in 1ml
physiological saline and then sperm count was performed
with help of Neubauer’s chamber under light
microscope.
Lipid Peroxidation: For the estimation of MDA level,
blood samples were centrifuged at 3000rpm for 110
minutes to collect serum. Level of MDA was determined
by standard TBARs method with slight modification by
UV spectrophotometer[12].
Estimation of Testosterone level: Testosterone level of
all groups of mice in the serum samples were determined
by using testosterone ELISA kit of LILAC Medicare (P)
ltd.
Statistical Analysis: Mean±SD and p-value were
obtained using SPSS software (Statistical Package for
Social Sciences, version 16.0). A p-value less than 0.05
were considered significant. Statistical analysis was
performed with the help of one way ANOVA.
RESULTS
Testis of the mice was weighed and significant changes
in the weight were observed upon endosulfan treatment.
The highest mean was observed in control group
(0.113g), followed by 4 weeks (0.101g), 8 weeks
(0.086g), 12 Weeks (0.075g) endosulfan treated mice
groups. Similarly, sperm count decreased noticeably
upon treatment with endosulfan in swiss albino mice.
Highest mean value of sperm count was recorded in
control group (5 million/µl) whereas mean sperm count
in endosufan treated mice were 0.412 million/µl (4
weeks treated mice group), 0.0938 million/µl (8 weeks
treated mice group), 0.0434 million/µl (12 weeks treated
mice group) with significance (p-value<0.0001).
Male testosterone level was detected highest in control
group (2.62 ng/ml) and 1.98 ng/ml, 1.83 ng/ml, and 1.22
ng/ml in endosulfan treated mice for 4 weeks, 8 weeks,
12 weeks respectively. Moreover, while assessing the
lipid peroxidation, the mean MDA level was measured
highest in endosulfan treated group for 12 weeks (46.62
nMol/ml), followed by 8 weeks (42.11 nMol/ml), 4
weeks (30.01nMol/ml) and control (22nMol/ml) with
significance (p-value<0.0001).
Fig 01: Testicular weight (a) and sperm count (b) in control and endosulfan treated mice for 4 weeks, 8 weeks,
and 12 weeks.

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Fig 02: Testosterone level (c) and MDA level (b) in control and endosulfan treated male mice for 4 weeks, 8
weeks and 12 weeks.
Table 01: Mean±S.D of Testicular weight, Sperm Count, Testosterone Level, MDA level of control and
endosulfan treated mice for 4 weeks, 8 weeks and 12 weeks.
Control
4 Weeks
8 Weeks
12 Weeks
Testicular weight (g)
0.113±0.055
0.101±0.042
0.086±0.029
0.075±0.027
Sperm Count
(Million/µl)
5±0.603
0.412±0.322
0.0938±0.027
0.0434±0.025
Testosterone level
(ng/ml)
2.626±1.198
1.98±0.98
1.83±0.855
1.22±0.596
MDA level (nMol/ml)
22.0±4.106
30.013±2.898
42.117±3.943
46.62±5.980
DISCUSSION
The present investigation reflects the changes in physio-
chemical mechanisms involved in reproductive system in
male mice due to administration of endosulfan. The
impairment of spermatogenesis in pubertal and
prepubertal rat induced by endosulfan has been
reported[13, 14]. The results clearly indicate that
endosulfan affects spermatogenesis when administered at
the rate of 2 mg/kg/body weight. It has been reported that
testicular weight, enzyme activity, and sperm count
significantly diminished in rats after the administration
of endosulfan at the rate of 2 mg/kg/body weight[15].
Similarly, our results demonstrate quite similar pattern of
derangement in the values of parameters like testicular
weight, sperm count etc. Endosulfan has also been
associated with the degeneration of seminiferous
epithelium, reduced number of leydig cells, and immotile
spermatozoa.
According to a study, endosulfan was observed to
increase the biotransformation of testosterone leading to
increased elimination through urine[16]. Our results shows
decrease in the testosterone level in male mice upon
endosulfan administration. Endosulfan has been
demonstrated to induce oxidative stress. It has been
reported that the endosulfan reduces the free radical
scavenging enzymes like SOD, Glutathione peroxidise,
catalases etc[17, 18]. Genotoxic and cytotoxic effects of
pesticides on plant tissue have been affirmed[19]. Higher
toxicity level is directly correlated with greater lipid
peroxidation, due to malfunction developed by
endosufan accumulated in cells.
However, effect of endosulfan on male reproductive
system needs to be studied extensively. Moreover, data
on enzyme activity, gene expression, and changes in
antioxidant system could unravel prominent features as
how endosulfan affects the reproductive system in male
mice.
CONCLUSION
Endosulfan acts as xenoestrogen and toxic agent. An
effort was made to study the effect of endosulfan of male
reproductive system. Endosulfan decreases the testicular
weight, spermatogenesis, testosterone level and increases
the oxidative stress. Endosulfan is still used in
agriculture of India widely and unrestrainedly.
Ultimately, endosulfan is consumed by humans
unknowingly which leads to the complications in
reproductive system.
ACKNOWLEDGEMENT
Authors greatly acknowledge financial assistance from
ICMR, New Delhi, Government of India. Authors are
indebted to Prof. M. Rajalakshami and Dr. R.S Sharma
for their kind support. We are thankful to S S Hospital
and Research Institute for providing infrastructure
facility.

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