Toxicology 200 (2004) 205211

Effects of endosulfan on B cells of Langerhans islets

in rat pancreas
Yusuf Kalender a, , Suna Kalender b , Meltem Uzunhisarcikli a ,
Ayse Ogutcu a , Fatma Aikgoz a , Dilek Durak c
a
c

Biology Department, Faculty of Arts and Science, Gazi University, 06500 Ankara, Turkey
b Biology Department, Faculty of Education, Gazi University, 06500 Ankara, Turkey
Biology Department, Yozgat Faculty of Arts and Science, Erciyes University, Yozgat, Turkey

Received 15 March 2004; received in revised form 31 March 2004; accepted 31 March 2004
Available online 10 May 2004

Abstract
Endosulfan is widely used in insect control and it is absorbed by both humans and animals through ingestion, inhalation, and
percutaneously. The purpose of this work was to study blood glucose levels and ultrastructural changes that might occur in the
pancreas of adult male Wistar rats as a result endosulfan intoxication. The treated group (n = 60) received endosulfan orally
via gavage 2.0 mg/kg per day in corn oil for 6 weeks, while the control group (n = 10) was given equal amount of corn oil for
the same period. The substances were administrated once a day. Blood glucose levels were significantly increased at the end of
3rd and 4th week (P < 0.05), and 5th and 6th week (P < 0.01) after administration of endosulfan to rats compared with the
control group. In electron microscopy studies, at the end of 2nd and 3rd weeks, swelling of mitochondria; at the end of 4th week,
vacuoles in cytoplasm; at the end of 5th week, dissolution of mitochondrial matrix; and at the end of 6th week, picnotic nucleus
in B cells in Langerhans islet were observed after endosulfan treatment.
2004 Elsevier Ireland Ltd. All rights reserved.
Keywords: Endosulfan; Pancreas; Langerhans islets; Glucose; Electron microscopy

1. Introduction
Pesticides are occasionally used indiscriminately in
large amounts causing environmental pollution, and
therefore, are a cause of concern. Residual amounts
of organochlorines (OC) and organophosphate (OP)
pesticides have been detected in the soil, water bodies,
Corresponding author. Tel.: +90-312-212-60-30x3047;
fax: +90-312-212-22-79.
E-mail address: kalender@gazi.edu.tr (Y. Kalender).

vegetables, grains, and other foods products (IARC,

1983; John et al., 2001). OC are known to cause inhibition of acetylcholinesterase activity in the target
tissues (Ansari et al., 1987; Dikshith et al., 1988). Toxicity of OP and OC pesticides causes adverse effects
on many organs (Tyagi et al., 1985). Other systems
that could be affected by OC intoxican are immune
system (Banerjee and Hussain, 1987), kidney (Singh
and Pandey, 1989), reproductive system (Sinha et al.,
2001), and pancreas (Hagar and Fahmy, 2002). Endosulfan is an organochlorine pesticide. Endosulfan

0300-483X/$ see front matter 2004 Elsevier Ireland Ltd. All rights reserved.
doi:10.1016/j.tox.2004.03.017

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Y. Kalender et al. / Toxicology 200 (2004) 205211

(6,7,8,9,10,10-hexachloro-1,5,5a,6,9,9a-hexahydro-6,
9 methano-2,4,3-benzodiexathiepin-3-oxide), a broad
spectrum insecticide of the cyclodiene group, is a central nervous system poison. It has been classified by
the WHO (1986) in the category of technical products
that are moderately hazardous. Endosulfan controls
a wide range of sucking and chewing insect pests
notably of the orders of Lepidoptera, Coleoptera, Heteroptera, Homoptera, Tysanoptera, Diptera, and some
species belonging to the order of Acarina (Naqvi and
Vaishnavi, 1993). It is especially used on non-food
crops such as cotton and tobacco and on food crops
such as vegetables, fruits, corn, cereals, tea, and coffee
(Naqvi and Vaishnavi, 1993).
The aim of the present study was to investigate effects of endosulfan on blood glucose level and ultrastructural changes in B cell in islets of Langerhans of
the rat pancreas.

to rats in experiment group. Control animals received

equal amount of corn oil. The substances were administered once a day in the morning to non-fasted rats.
All rats were treated for 6 weeks.
2.4. Measurement of blood glucose levels
At the beginning of the experiment, glucose levels were measured from the tail vein of control group
rats. After administration of endosulfan, blood glucose was measured at weeks 16 with a glucometer
(Lifescan, Johnson Co., One Touch Basic, USA) in
all animals. Rats were anesthetized with ether. Blood
samples taken from the heart were collected into heparinized tubes and then pancreas tissues were taken
for electron microscopic examinations.
All results were expressed as mean S.D. and analyzed using Students t-test.
2.5. Electron microscopy studies

2. Material and methods

2.1. Animals
Male Wistar rats (weighing approximately 150
180 g), obtained from the Refik Saydam Central
Hygiene Institute, Ankara, Turkey, were used. The
animals were housed in plastic cages, fed a standard
laboratory diet (containing 28.0% of carbohydrates,
23.5% of proteins, 4.0% of fat, 3.5% of fibrous matter, and was supplemented with vitamins and trace
elements) and water ad libitum, and treated in accordance with the guidelines of the Animal Care
Committee of the Gazi University, Turkey.
2.2. Chemical
Endosulfan, technical purity 95%, was obtained
from Refik Saydam Hfzsshha Institute, Poison Research Center, Ankara, Turkey.

For electron microscopic examinations of pancreas,

primer fixation was made in 3% glutaraldehyde (Agar
Sci. Ltd., Essex, England) in sodium phosphate buffer
(200 mM, pH 7.4) (Merck, Alfred Paluka Co., Turkey)
for 3 h at 4 C. Materials were washed with the same
buffer and postfixed in 1% osmium tetroxide (Agar
Sci. Ltd., Essex, England) and in sodium phosphate
buffer (pH 7.4) for 1 h at 4 C. Tissue samples were
washed with the same buffer for 3 h at 4 C, and were
dehydrated in graded ethanol series (Agar Sci. Ltd.,
Essex, England) and were embedded in Araldite (Agar
Sci. Ltd., Essex, England). Thin sections were cut with
Reichert OM U3 (Leica Co., Austria) ultramicrotome.
Samples were stained with 2% uranyl acetate and lead
citrate. The sections were viewed and photographed
on a Jeol 100 CX II transmission electron microscope
(TEM) (Jeol Ltd., Japan) at 80 kV.

3. Results
2.3. Animal treatment schedule
3.1. Evaluation of blood glucose levels
Endosulfan treatments to rats were done according
to Sinha et al. (2001). Rats were divided into two
groups, control (n = 10) and experiment groups (n =
60). Endosulfan at a dose of 2.0 mg/kg per day in
corn oil (0.2 ml per animal) was given through gavage

No death was observed in any of the experimental

groups. The administration of endosulfan for 1 and
2 weeks produced no significant increase in blood
glucose level compared with the control group (P >

Y. Kalender et al. / Toxicology 200 (2004) 205211

207

Table 1
The effect of oral administration of endosulfan (2 mg/kg per day) on blood glucose levels of the male Wistar rats
Groups

Control
Endosulfan
P

< 0.05 and

Glucose (mg/dl) (mean S.D.)

Week 1

Week 2

Week 3

Week 4

Week 5

Week 6

80.3 7.55
82.4 6.48

79.1 7.43
85.3 6.87

79.3 6.49
96.6 7.50

80.1 6.31
99.7 6.43

79.7 7.07
114.5 8.11

79.3 6.86
115 7.24

< 0.01 compared to control group.

0.05). In endosulfan-treated rats significant increase in

blood glucose level compared with the control group
was observed at the end of 3rd and 4th weeks (P <
0.05), and 5th and 6th weeks (P < 0.01) (Table 1).
3.2. Evaluation of the results of electron
microscopy
Electron microscopic analysis of B cells in Langerhans islet showed no pathological changes in control
group. B cells contained number of secretory granules. The granules were composed of a central core,
usually of moderate homogenous or slightly heterogeneous electron density, and an external single-layered
membrane. The granules had a space between the core
and the membrane. The granules were diffusely distributed in the cytoplasm (Fig. 1). In addition, B cells
in Langerhans islet had round nucleus and mitochon-

dria were abundant in cytoplasm (Fig. 1). One week after endosulfan treatment to rats no pathological change
was observed in B cells in Langerhans islet (Fig. 2).
Swelling of some mitochondria was observed in B
cells after 2 weeks (Fig. 3). At the end of the 3rd and
4th weeks swelling of some mitochondria and dissolution of mitochondrial matrix in B cells were observed
(Figs. 4 and 5). Also, at the end of the 4th week dilation of endoplasmic reticulum was observed (Fig. 5).
Some B cells in Langerhans islet of the pancreas had
weak picnotic nucleus at the end of 5th week. Large
and small vacuoles occurred in cytoplasm. Especially
neighboring empty secretory granules connected to
each other and then produced vacuoles (Fig. 6). Six
weeks after endosulfan treatment granule number decreased. Vacuoles produced by granules in cytoplasm
were increased. In this stage, picnotic nucleus in B
cells was observed (Fig. 7).

Fig. 1. Electron micrograph of B cell in Langerhans islet of control group rat pancreas. N: nucleus, M: mitochondria, G: Golgi complex,
secretory granules (arrows) 13,500.

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Y. Kalender et al. / Toxicology 200 (2004) 205211

Fig. 2. Electron micrograph of B cell of Langerhans islet 1 week after endosulfan treatment, showing no pathological change in rat
pancreas. N: nucleus, M: mitochondria, endoplasmic reticulum (white arrows), secretory granules (black arrows) 13,500.

Fig. 3. Electron micrograph of the swelling of mitochondria (M) in B cells of Langerhans islet in rat pancreas 2 weeks after endosulfan
treatment. N: nucleus, secretory granules (arrows) 13,500.

4. Discussion
Endosulfan is absorbed by both humans and animals through inhalation, ingestion, and percutaneously. The gastrointestinal tract is also a site for
absorption of endosulfan following ingestion (Naqvi
and Vaishnavi, 1993). The oral LD50 values for
technical endosulfan ranges from 7 to 121 mg endo-

sulfan/kg depending upon species, sex, formulation

tested, vehicle used, and nutritional status of the animal (WHO, 1984; Naqvi and Vaishnavi, 1993). The
oral LD50 of endosulfan in rats for the alpha-isomer
is 76 and 240 mg/kg for the B-isomer (Maier-Bode,
1968). Gupta and Chandra (1977) administered
10 mg endosulfan/kg per day to rats by gavage. The
chemical was given in peanut oil for 15 days, which

Y. Kalender et al. / Toxicology 200 (2004) 205211

209

Fig. 4. Electron micrograph of the swelling of mitochondria (M) and dissolution of mitochondrial matrix in B cells 3 weeks after endosulfan
treatment. N: nucleus, secretory granules (arrows) 16,000.

Fig. 5. Electron micrograph of the swelling of mitochondria (M) and dissolution of mitochondrial matrix and dilation of endoplasmic
reticulum (ER) in B cells 4 weeks after endosulfan treatment. N: nucleus, secretory granules (arrows) 13,500.

caused inflammation of the lungs and dilated alveoli.

In this study even though endosulfan was given under LD50 dose, we observed pathological findings in
B cells.
Chronic endosulfan administration for 2 months led
to degenerative changes of various degrees of the pancreatic islets as well as the exocrine acini. The de-

generation of the acini might be considered as a sort

of toxic inflammatory process simulating pancreatitis (Marsh et al., 1988). Although we didnt mention
exocrine acini we observed pathological findings in
them.
Insufficiency of insulin hormone causes the increase of blood glucose level. In this study, especially

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Y. Kalender et al. / Toxicology 200 (2004) 205211

Fig. 6. Electron micrograph of the vacuoles (V) and connecting of neighboring empty secretory granules (arrows) in cytoplasm of B cells
and picnotic nucleus (N) 5 weeks after endosulfan treatment 13,500.

Fig. 7. Electron micrograph of increase of vacuoles (V) in cytoplasm of B cell and picnotic nucleus (N) 6 weeks after endosulfan treatment
10,000.

at the end of the 5th and 6th weeks, blood glucose

level significantly increased. This shows that B cells
couldnt secrete adequate insulin. Actually some insecticides cause ultrastructural changes also in A cells
of Langerhans islet (Hagar and Fahmy, 2002; Naqvi
and Vaishnavi, 1993). Biochemical studies show that
endosulfan affects integral proteins and receptors of
cell membrane (Kiran and Varma, 1990; Singh and

Pandey, 1989). Hagar and Fahmy (2002) reported that

dimethoate produced the decrease of insulin level in
rats. We didnt measure insulin hormone level. Even
if adequate levels of insulin hormone was secreted,
there will be degeneration of receptors of target
cells membranes, so this will cause the increase of
blood glucose level. Garg et al. (1980) observed that
blood glucose level increased after oral endosulfan

Y. Kalender et al. / Toxicology 200 (2004) 205211

administration and Hagar and Fahmy (2002) observed

the increase of blood glucose level after dimethoate
administration, which is an organophosphorus insecticide. Our results are in agreement with them.
In this study endosulfan caused vacuoles and
swelling of mitochondria. According to these, the decrease of mitochondrial enzyme activity and calcium
level might be observed. Moussa and Hafez (1995)
showed that dimethoate inhibited mitochondrial enzyme activity.
After administration of endosulfan for 4 weeks we
observed picnotic nucleus in B cells of the Langerhans islets. While at the end of the 4th week, weak
picnotic nucleus was observed, at the end of the 6th
week, extremely picnotic nucleus was observed in B
cells of Langerhans islets. Picnotic nucleus is seen especially in irreversible damage. It can be observed as
a result of the increase of calcium level in cell. In this
study vacuolization of mitochondria and dilation of
endoplasmic reticulum showed that calcium ion concentration in cell was damaged.
Blood glucose levels and ultrastructural changes in
this study show that endosulfan affects B cells of pancreas in rats even if it is under the LD50 dose level. Endosulfan not only has toxic effects on mammalian and
other animals but it causes pollution as well. Therefore, microbial insecticides which are effective on target organisms and dont cause pollution should be used
instead of endosulfan.

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