Download Effect of Chlorpyrifos on Thyroid Gland of Adult Male

Egypt. J. Histol. Vol. 33, No. 3, Sep., 2010: 441 - 450
(ISSN: 1110 - 0559)
Original Article
Effect of Chlorpyrifos on Thyroid Gland of Adult Male Albino Rats
Abeer M. Shady1 and Fayroz I. Noor El-Deen2
1
Histology and 2Forensic and Toxicology Departments, Faculty of Medicine, Menoufyia University
ABSTRACT
Introduction: Chlorpyrifos (CPF) is the most widely used organophosphorus insecticide.
Aim of the Work: This work aimed to clarify effects of repeated doses of chlorpyrifos on thyroid gland of adult male
albino rats.
Materials and Methods: Fifteen adult male rats were used. They were divided into two groups: control and chlorpyrifos
treated. On sacrifice, blood samples were drawn to determine serum T3, T4 and TSH levels. Thyroid glands were
processed for histological, histochemical, immunohistochemical and morphometrical studies.
Results: Chlorpyrifos treated rats showed a highly significant decreased body weight, decreased thyroid weight,
decreased T3 and T4 and increased TSH hormone levels. Histologically, there was decrease in the size of follicles
and amount of colloid, focal degeneration of follicular cells, thickened collagen fibers and congested blood vessels.
Histochemically, there was decrease in the PAS reaction. Immunohistchemically, there was a strong caspase 3 and a
weak thyroglobulin protein expressions. Ultrastructurally, treated rats had small heterochromatic nuclei, degenerated
mitochondria and few secretory granules. Morphometrically there was a highly significant decreased size of thyroid
follicles, decreased area % of colloid and increased area % of collagen fibers.
Conclusion: CPF could seriously affect thyroid glands with decreased thyroid hormones which play an important role
in most of body physiological processes. Use of CPF and other insecticides should be extremely limited and be under
many precautions.
Key Words: Chlorpyrifos, thyroid gland, rats, caspase-3,
thyroglobulin.
Corresponding Author: Abeer M. Shady
Tel.: 0111579691
E-mail: [email protected]
INTRODUCTION
hormonal alterations12 as it is considered as a potential
endocrine disrupter13. The most commonly affected
endocrine gland is the thyroid as it is considered as a
sensitive target to CPF leading to long-term effects on
thyroid function14. Effects on thyroid function have
attracted lots of attention because thyroid hormones act
on cells of almost all tissues and therefore are involved
in several physiological processes during life span15. In
adults, thyroid hormones are involved in metabolism of
protein, lipid and carbohydrate and in heat generation16.
They are also necessary for normal reproductive
functions, regulation of heart rate and gastrointestinal
motility, as well as for emotional stability17. Disruptions
of thyroid function by endogenous or exogenous factors
may produce various subclinical effects18 or direct
clinical manifestations19.
Organophosphorus (OP) insecticides are extensively
used for control of insects in home and agricultural
practice1. They comprise over half of all insecticide
used worldwide and employed in domestic, agricultural
and non-agricultural (i.e. schools, golf courses, parks)
settings2. However many OP insecticides have been
removed from home usage due to their potential
for neurotoxicity in children, but they are still used
agriculturally on crops such as cotton, corn, almonds,
oranges and apples3 for achieving better quality products4.
Chlorpyrifos (CPF) with a trade name of Dursban is the
most widely used OP insecticide5. Exposure to CPF is
known to cause millions of acute poisoning allover the
world1. Besides acute poisoning, long-term and repeated
low dose exposure to CPF could cause many chronic
effects on different organs6. It could lead to disruptive
effects on CNS development and behavior7. Chlorpyrifos
could induce severe testicular damage and results in
reduction of sperm count and thus affect fertility8. It may
have also toxic effects on liver4, thymus9 or heart10 and
it possibly has increased risk of testicular, prostate or
thyroid cancer11. Chlorpyrifos may also cause long-term
AIM OF THE WORK
Was to clarify the effect of repeated doses of chlorpyrifos
on thyroid gland of adult male albino rats.
39 (1204-2010)
441
Abeer M Shady and Fayroz I Noor El-Deen
MATERIALS AND METHODS
B. Histochemical Study:
Chemicals:
Five μm paraffin sections were stained with periodic
acid Schiff (PAS) reaction for mucopolysaccharides24.
Chlorpyrifos is a colorless crystals obtained from
El-Gomhouria Company for Trading Chemicals and
Medical Appliances. It was dissolved freshly in corn oil
for a final concentration of 0.5 mg/ml.
C. Immunohistochemical Study:
Five μm paraffin sections were incubated with either
a monoclonal anti- rat caspase-3 antibody (Lab vision,
USA) in a dilution of 1:50025 for detection of apoptosis
or with a monoclonal anti human thyroglobulin (Dako,
Japan) (which has a cross reactivity with thyroglobulin
from mouse and rat) in a dilution of 1:10026 for
demonstration of colloid using the avidin biotin
peroxidase method27.
Animals Used:
Fifteen adult male albino rats weighing 200-225 gm
were used in this study. They were allowed for laboratory
rat chow diet and water ad-libitum. Strict care and
hygiene were taken to maintain a normal and healthy
environment for all rats all time.
III. Electron Microscopic Study:
Experimental Design :
Small pieces of thyroids were rapidly fixed in 3%
glutaraldehyde and 0.1 M phosphate buffer at pH 7.4,
postfixed in osmium tetraoxide and embedded in epon.
Semithin sections (1u.m thick) were prepared and stained
with toluidine blue. Ultrathin sections (50-80 nm thick)
were stained with uranyl acetate and lead citrate and
examined by Philips transmission electron microscope28.
Rats were randomly divided into 2 groups as follow:
Group I (Control):
Five rats, two of them were kept without treatment.
The other three were given 2 ml of corn oil once daily
orally by a modified plastic syringe for 14 days.
IV. Morphometrical Study:
GroupII (Chlorpyrifos Treated):
Central regions of thyroid glands from all rats were
examined under light microscopy using an objective lens
of magnification 40 by using the interactive measuring
menu of image analyzer (Lecia Qwin 500 image analyzer
computer system, England). Size of thyroid follicles (by
measuring their areas) was measured in H and E sections,
area % of colloid was measured in PAS-stained sections
and area % of collagen fibers was measured in Masson
trichrom-stained sections. These measurements were
done in 10 fields of each specimen.
Ten rats were given chlorpyrifos once daily orally by
a modified plastic syringe in a dose of 5 mg / Kg body
weight20 which equals 1/50 of LD5021 for 14 days22.
Sampling, Sectioning and Staining:
Twenty-four hours after the last dose, rats were
weighed and anaesthetized by intraperitoneal injection of
sodium pentobarbital in a dose of 50 mg/kg23 to obtain
blood samples from eye balls by capillary tubes then they
were sacrificed by decapitation.
V. Statistical Analysis:
1. Biochemical Study:
Data were expressed as means ± SD. The results were
computed statistically (SPSS for Windows, Version 14.0,
Chicago, IL, U.S.A.) using student t-test for comparison
between two groups of normally distributed variables
to compare experimental group with the control. The
0.05, 0.01 and 0.001 probability was used as criterion
of significance, highly significance and very highly
significance, respectively.
Blood samples were analyzed to determine serum triiodothyronin (T3); tetra-iodothyronin (T4) and thyroid
stimulating hormone (TSH) levels.
II. Light microscopic Study:
A. Histological Study:
RESULTS
Skin of the neck was incised and trachea was
removed. Thyroid gland on the posterior aspect of
trachea was removed. They were washed in saline, dried
by a filter paper and weighed. Then they were fixed in
10% neutral buffered formol and processed for paraffin
blocks. Five μm paraffin sections were cut and stained
with hematoxylin and eosin (H and E) stain for routine
histological study and Masson trichrome for collagen
fibers24.
I. General Results:
Chlorpyrifos treated rats showed a very highly
significant decrease in body weight and a highly
significant decrease in thyroid weight when compared
with the control (Table 1 and Diagrams 1).
442
Effect of Chlorpyrifos on Thyroid Gland of Adult Male Albino Rats
Table I: Body and thyroid weights in control and
experimental groups.
Body weight in gm
Thyroid weight in mg
Group of
animals
Mean ±SD
P value
Mean±SD
P value
Control
212 ± 14
--------
17.77 ± 0.19
-------
Chlorpyrifos
treated
167 ± 9
< 0.001
15.15 ± 0.15
<0.01
II. Biochemical Results:
Diagram 1: Body and thyroid weights in control and experimental
groups.
Chlorpyrifos treated rats showed a very highly
significant decrease in serum Tri-iodothyronin (T3) and
Tetra- iodothyronin (T4) and a very highly significant
increase in thyroid stimulating hormone (TSH) levels
when compared with the control (Table 2 and Diagram 2).
Table 2: Serum hormone levels in control and experimental groups.
T3 in μg/100 ml
T4 in μg/100 ml
TSH in μg/100 ml
Group of animals
Mean+SD
P value
Mean + SD
P value
Mean + SD
P value
Control
0.47 ± 0.06
--------
5.10 ± 0.61
---------
10.28
---------
Chlorpyrifos treated
0.36 ± 0.11
<0.001
3.33 ± 0.23
<0.001
13.51
< 0.001
Diagram 2: Hormonal assay in control and experimental groups.
III. Light Microscopic Changes:
marked thyroglobulin protein expression in the colloid
(Fig. 5) and a weak cytoplasmic caspase-3 protein
expression in follicular cells (Fig. 6).
Thyroid sections of control rats showed thyroid
follicles of various sizes which were lined mainly by
cubical follicular cells with rounded vesicular nuclei and
few parafollicular C cells. They contained on their centers
acidophilic homogenous colloid. Between follicles,
there were interfollicular cells and blood capillaries
(Figs. 1, 2). The follicles were separated by thin collagen
fibers (Figs. 3). Basal lamina of follicular cells showed
moderate PAS reaction while colloid showed a marked
reaction (Fig. 4). Immunohistochemically, there was a
Sections of treated rats showed apparently decrease in
the size of thyroid follicles and amount of colloid. Some
follicles showed vacuolated colloid while others were
devoid of colloid. Some follicular cells were degenerated
having shrunken nuclei and vacuolated cytoplasm. Some
follicles had exfoliated cells in their lumens. Interfollicular
tissue showed dilated congested blood vessels and focal
interstitial hemorrhage (Figs. 7-9). Collagen fibers between
443
Abeer M Shady and Fayroz I Noor El-Deen
the follicles were thickened (Fig. 10). Basal lamina of
follicular cells showed a weak PAS reaction while colloid
showed a moderate reaction (Fig. 11).
Some follicular cells of treated rats showed small
nuclei with irregular outlines and abnormal distribution
of chromatin. Their cytoplasm showed large vacuoles
occupying most of the cells with dilated cisternae of
RER, degenerated mitochondria and few secretory
granules (Fig. 15).
Immunohistochemically, there was a weak
thyroglobulin protein expression in the colloid (Fig. 12)
and a marked cytoplasmic caspase 3 protein expression
in follicular cells (Fig. 13).
V. Morphometrical Study and Statistical Analysis:
Morphometrical study confirmed the light
microscopic results and revealed a highly significant
decreased size of thyroid follicles, decreased area % of
colloid and increased area % of collagen fibers (Table 3
and Diagram 3).
IV. Electron Microscopic Changes:
Follicular cells of control rats appeared with rounded
euchromatic nuclei and numerous short apical microvilli.
Their cytoplasm contained dilated RER, abundant
mitochondria, prominent Golgi apparatuses, SER,
lysosomes, abundant apical vesicles containing colloid
and electron dense granules (Fig. 14).
Table 3: Size of follicles and % of colloid and collagen.
Size of follicles
Area % of colloid
Area % of collagen
Group of animals
Mean + SD
P value
Mean + SD
P value
Mean + SD
P value
Control
3663.71 ± 35
-------
21.34±0.93
------
1.5213 ±0.12
------
CPF treated
1975.63 ± 22
< 0.001
10.16±0.63
< 0.001
4.7612 ±0.59
< 0.001
Diagram 3: Morphometrical study of control and experimental groups.
444
Effect of Chlorpyrifos on Thyroid Gland of Adult Male Albino Rats
Fig. 1: Thyroid gland of a control rat showing thyroid follicles of various
sizes lined mainly with follicular cells (arrows) and having homogenous
acidophilic colloid (C). Notice: interfollicular tissue (IF). H and E X 200
Fig. 4: Thyroid gland of a control rat showing marked PAS reaction in the
colloid (C) and a moderate reaction in the basement membrane (arrows).
PAS X 400
Fig. 2: Thyroid follicles of a control rat showing follicular cells having
vesicular nuclei with prominent nucleoli (arrows). Notice: homogenous
colloid (C).
Touilidine blue X 1000
Fig. 5: Thyroid gland of a control rat showing strong immunohistochemical
thyroglobulin protein expression in the colloid (C).
PAP X 200
Fig. 3: Thyroid gland of a control rat showing thin collagen fibers between
thyroid follicles (arrows).
Masson trichrome X 400
Fig. 6: Thyroid gland of a control rat showing weak immunohistochemical
caspase-3 protein expression in cytoplasm of follicular cells. PAP X 200
445
Abeer M Shady and Fayroz I Noor El-Deen
Fig. 7: Thyroid gland of a chlorpyrifos treated rat showing thyroid
follicles lined with vacuolated follicular cells (red arrows) and having
vacuolated colloid (V). Some follicles are very small in size (F) Notice:
Dilated congested blood vessels (BV).
H and E X 200
Fig. 10: Thyroid gland of a chlorpyrifos treated rat showing thick collagen
fibers between thyroid follicles (arrows). Notice: Dilated congested blood
vessels (BV).
Masson trichrome X 400
Fig. 8: Thyroid gland of a chlorpyrifos treated rat showing some
follicles with exfoliated cells in their lumens (arrows). Notice: interstitial
haemorrhage (arrows).
H and E X 200
Fig. 11: Thyroid gland of a chlorpyrifos treated rat showing moderate PAS
reaction in the colloid (C) and a weak reaction in the basement membrane
(arrows).
PAS X 400
Fig. 9: Thyroid follicles of a chlorpyrifos treated rat showing some
follicular cells having vesicular nuclei with prominent nucleoli (thick
arrows). Others have shrunken nuclei (thin arrows). Notice: scanty lightly
stained colloid (C) and thick collagen fibers between follicles (S).
Touilidine blue X 1000
Fig. 12: Thyroid gland of a chlorpyrifos treated rat showing weak
immunohistochemical thyroglobulin protein expression in the colloid.
PAP X 200
446
Effect of Chlorpyrifos on Thyroid Gland of Adult Male Albino Rats
Fig. 13: Thyroid gland of a chlorpyrifos treated rat showing marked
immunohistochemical caspase-3 protein expression in cytoplasm of
follicular cells (arrows).
PAP X 200
Fig. 16: Follicular cells of a chlorpyrifos treated rat showing eccentric
heterchromatic nuclei with irregular outlines (N) and large vacuoles
occupy most of cells (V). Notice: thick collagen fibers (CF). X 8000
Fig. 14: Follicular cells of a control rat showing rounded euchromatic
nuclei with smooth outlines (N) and numerous short apical microvilli
(M). There is a prominent infranuclear Golgi apparatus (G), dilated RER
(ER) and abundant mitochondria (arrow).
X 8000
Fig. 17: Follicular cell of a chlorpyrifos treated rat showing some intact
mitochondria (M) and others are degenerated (m).
X 16000
DISCUSSION
Little Chlorpyrifos is a widely used organophosphorus
insecticide and considered as potential endocrine
disrupters7. Its Effects on thyroid function have attracted
lots of attention because thyroid hormones play a major
role in mammalian physiological processes4.
Chlorpyrifos treated rats showed significant decrease
in their body weight and thyroid weight. This agreed with
the findings of other researchers18,29. Decreased body
weight could be referred to systemic intoxication29 due
to cholinesterase inhibition with subsequent cholinergic
overstimulation2; marked DNA damage30 or altered
carbohydrate metabolism31. Decreased thyroid weight
might be owed to decreased colloid contents in the
follicles or to degenerated follicles.
Fig. 15: Follicular cell of a control rat showing apical microvilli (M),
secretory vesicles containing colloid (C), dilated rough endoplasmic
reticulum (RER) and intact mitochondria (m).
X 16000
447
Abeer M Shady and Fayroz I Noor El-Deen
Chlorpyrifos treated rats showed hypothyroidism
which was evidenced biochemically by a very highly
significant decrease in serum T3 and T4 levels,
histologically by decreased amount of colloid,
histochemically by decreased PAS reaction in the colloid
and Immunohistochemically by a weak thyroglobulin
protein expression in the colloid. Thyroglobulin is
the glycoprotein precursor of the iodinated thyroid
hormones thyroxine (T4) and triiodothyronine (T3)32.
It is synthesized by the follicular cells and stored in the
lumen of the follicles33. It exhibits the general properties
of the globulins32.
3.
4.
5.
6.
Hypothyroidism in chlorpyrifos treated rats was
reported also by some investigators7,34 who referred it
to degeneration and apoptosis of follicular cells with
subsequent decrease in their secretion35.
7.
Degeneration and apoptosis of follicular cells was
noticed in chloropyrifos treated rats as evidenced
histologically by shrunken nuclei and vacuolated
cytoplasm, ultrastructurally by small heterochromatic
nuclei with irregular outlines and degenerated
mitochondria and Immunohistochemically by a strong
caspase 3 protein expression. Caspase-3 is a member
of interlukein converting enzymes. It is the most
commonly one involved in the execution of apoptosis
in various cell types. Increase reactivity for caspase-3
could indicate cellular apoptosis36. Previous studies
reported also degeneration and apoptosis of follicular
cells30,34. This might be owed to oxidative damage caused
by chlorpyrifos with generation of Reactive Oxygen
Species (ROS)37 and lipid peroxidation30. Thickened
collagen fibers between thyroid follicles might be related
to Lipid peroxidation which could accelerate collagen
synthesis by stimulating stellate cells38. Congested blood
vessels could be referred to oxidative stress and lipid
peroxidation which might affect vascular walls leading
to their dilatation and congestion. From the foregoing it
is clear that CPF could seriously affect thyroid gland with
subsequent decreased thyroid hormones which play an
important role in most of body physiological processes
and can regulate and maintain normal physical, mental
and sexual activities. So use of all insecticides and
especially chlorpyrifos should be extremely limited and
be under many precautions with further research about
more safe methods against insects.
8.
9.
10.
11.
12.
13.
14.
15.
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449
‫‪Abeer M Shady and Fayroz I Noor El-Deen‬‬
‫الملخص العربى‬
‫تأثير الكلوربيريفوس على الغدة الدرقية لذكور الجرذان البيضاء البالغة‬
‫عبير محمد شادي‪ ،1‬فيروز ابراهيم نور الدين‬
‫‪2‬‬
‫‪1‬قسمي الهستولوجيا و ‪2‬الطب الشرعي و السموم ‪ -‬كلية الطب ‪ -‬جامعة المنوفية‬
‫مقدمة البحث‪ :‬يعتبر الكلوربيريفوس من أكثر المبيدات الحشرية استخداما‪.‬‬
‫الهدف من البحث‪ :‬لقد صمم هذا البحث لتوضيح آثار الجرعات المتكررة من الكلوربيريفوس على الغدة الدرقية لذكور الجرذان‬
‫البيضاء البالغة‪.‬‬
‫المواد المستخدمة و الطرق المتبعة‪ :‬لقد أجريت هذه الدراسة على خمسة عشر من ذكور الجرذان البيضاء البالغة والتي‬
‫قسمت عشوائيا إلى مجموعتين استخدمت األولى كمجموعة ضابطة‪ ‬أما الثانية فتم معالجتها بالكلوربيريفوس‪ .‬و عند الذبح‬
‫تم اخذ عينات دم لمعرفة مستوى هرمونات‪ T4 ,T3‬و ‪ TSH‬ثم أخذت الغدد الدرقية و استخدمت للدراسة الهستولوجية‬
‫بصبغتي الهيماتوكسيلين وااليوسين و الماسون ثالثي األلوان و لدراسة التركيب الدقيق بالمجهر اإللكتروني و كذلك للدراسة‬
‫الهستوكيميائية بصبغة الشيف الحامضية و للدراسة الهستوكيميائية مناعية لوجود الثيروجلوبلين و الكاسبس ‪ .3‬كما تم عمل‬
‫دراسة قياسية شكلية وجداول إحصائية لحجم الكريات و نسبة مساحة المنطقة الغروانية و نسبة مساحة ألياف الكوالجين‪.‬‬
‫النتائج‪ :‬لقد لوحظ وجود نقص بالغ الداللة في وزن الجسم و وزن الغدة الدرقية و كذلك في مستوى هرمونات‪T4 ,T3‬‬
‫و ‪ TSH‬في الدم في الجرذان المعالجة بالكلوربيريفوس كما أظهرت النتائج وجود انكماش في حجم الكريات مع تحلل للخاليا‬
‫و الميتوكوندريا وتمدد للشبكة اإلندوبالزمية الخشنة مع قلة مساحة المنطقة الغروانية و زيادة ألياف الكوالجين و وجود العديد‬
‫من األوعية الدموية المحتقنة‪ .‬كما كان هناك انخفاض في تفاعل الشيف الحامضي و الثيروجلوبلين مع زيادة في الكاسبس ‪.3‬‬
‫الخالصة‪ :‬مما سبق يتضح أن الكلوربيريفوس من الممكن أن يؤثر تأثيرا خطيرا على الغدة الدرقية مما يؤدي إلى انخفاض‬
‫هرموناتها و التي تلعب دورا هاما في معظم العمليات الفيسيولوجية بالجسم‪ .‬ولذلك ينبغي ان يكون استخدام المبيدات الحشرية‬
‫محدود للغاية وتحت العديد من االحتياطات‪.‬‬
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