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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. REFERENCES 1. 2. Tan DH, Peng SQ, Wu YL, Wang YM, Lu CF, Ding W, Wang QX and Yan CH. (2009): Chlorpyrifos induces delayed cytotoxicity after withdrawal in primary hippocampal neurons through extracellular signal-regulated kinase inhibition. Biol. Pharm.Bull. ;32(10):1649-1655. Roegge CS, Timofeeva OA, Seidler FJ, Slotkin TA and Levin ED. 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World J.Gastroenterol. ;15(12):1452-1458. 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. 450