WASTING SYNDROME AS A RESULT OF THE EXPOSURE OF ALBINO RATS TO 2,3,7,8-TETRACHLORODIBENZO-P-DIOXIN (TCDD)

M. Abd El-Nasser; A. Shehata; Eman, E. El-Sharkawy and D. A. Salem

Dept. of Forensic Medicine and Toxicology, Faculty of Veterinary Medicine, Assiut University.

INTRODUCTION :

                Exposure to TCDD may occur through diet especially food from animal origin or from the surrounding environment like soil, water and air as a result of bioaccumulation through food chain (Kreuzer et al, 1997). Also occupational exposure through industrial activities was occurred (WHO,1994). Changes in carbohydrates and lipid metabolism are very prominent symptoms of high doses of TCDD, resulting mostly in the loss of fat and muscle. The sever form of body-weight loss are described as "Wasting syndrome", the loss of body weight up to 50% is not entirely attributable to the lack of food intake. The explanation of a drastic TCDD-mediated decline in the adipose lipoprotein lipase activity is considered to induce lipolysis in adipose cells and increase in serum lipids as increase cholesterol and triglycerides levels. The wasting syndrome is lethal and develops over a period of a few week (2-3 weeks) in rat. TCDD produced changes in lipid metabolism associated with increase of rat serum cholesterol level (Mitrou et al., 2001). Rats administered 2.5, 25, 250 or 1000 ng TCDD/kg b.w/day, showed an increase of cholesterol level (Chu et al., 2001). TCDD in a dose of 0.01, 0.1, 1.0 and 3.0µg/kg b.w/day for 91 days revealed an increase in triglycerides levels in all doses in rat (Ivens et al.,1993). TCDD administration induced highly increase in lipid concentration in serum including increase of triglycerides levels (Mitrou et al., 2001). In monkeys TCDD elevated levels of serum triglycerides (Rier et al., 2001). In fact progressive hypoglycemia has been suggested as ultimate cause of TCDD-induced death (Gorski et al., 1990). Additional studies have revealed reduced utilization of glucose in Sprague-Dawely rats as early as 1 day after exposure to a lethal dose of TCDD (Weber et al., 1987). TCDD administered to male and female rats for 91 days in a dose of 0.01,0.1,1.0 and 3.0 µg/kg b.w by gavage showed a reduction of serum T4 levels in all doses (Ivens et al., 1993 and Battershill, 1994). TCDD induced lethality may be due to feed refusal, body weight loss and some other metabolic disorders. The objective of this study was to investigate the relation between TCDD application and the appearance of wasting syndrome. Investigation of the mechanism by which TCDD induces wasting syndrome was also our goal of this study.

MATERIALS AND METHODS:

Chemicals:

                TCDD solution (99% purity), was obtained from Grey Hound Company Laboratories, England. It was dissolved in corn oil as vehicle.

Experimental protocol:

                200 males albino rats weighting 100-150g of 10-12 weeks old were housed in wire bottom galvanized cages, five rat each. Food and water were provided ad libitum. Suitable temperature under 12 h. light/dark cycle was also provided. Forty rats were exposed to oral single dose of 4.4µg/kg b.w diluted in 1ml corn oil, while another forty rats were used as control group for the acute toxicity. Forty rats were exposed to 0.44µg/kg b.w, diluted in 1ml corn oil orally day by day for 12 weeks. Forty rats were exposed to the same previous dose in addition to Vit. C in concentration of 1g/l drinking water simultaneously. Forty rats were administered corn oil only and kept as control for long-term toxicity experiment. Samples were collected at 12, 24, 48, 72, 96 and 144 hours post-exposure in case of acute toxicity, while samples were collected after 4, 6, 8, 10, 12weeks post exposure and after 2, 4 weeks from the stoppage of TCDD exposure in the long term toxicity experiment. Average food consumption as well as body weight gain were recorded weekly. serum glucose concentration and triglycerides were determined colorimetrically according to the method of Trinder (1969). Cholesterol was estimated colorimetrically after the method of Stein (1986). Thyroxin level was determined in serum using enzyme linked immunoassay (ELISA) according to Kaplan (1997). Statistical analysis of data was conducted using SAS Institute statistical package (1990).

RESULTS:

                Body weight gain was severely impaired by 0.44µg/kg of TCDD in the time-course experiment as presented in table (1); Compared to control rats the difference was statistically significant from the second week to the last week after treatment. Rats displayed signs of wasting from the second week of treatment. Reduction of feed intake was also recorded after second week of TCDD treatment and persisted to the end of the experiment as shown in table (1). Rats displayed signs of wasting syndrome from the second week of exposure.

Table (1): Effect of long-term exposure of TCDD and TCDD + Vit. C on body weight and feed intake of albino rats.

* Significant at P  < 0.05 to 0.01 in comparison with control.

The letter  a  indicates significant differences between Vit. C-treated and non treated groups.

                Increase in both cholesterol and triglycerides levels in serum leading to hyperlipaemia, while it causes decrease in both glucose and thyroxin (T4) levels in serum leading to hypoglycemia and hypothyroidism as recorded in tables (2 & 3).

Table (2): Acute toxic effect of TCDD exposure on cholesterol, triglycerides, glucose and Thyroxine hormone (T4) levels of albino rats.

* Significant at P <0.05 to 0.01 in comparison with control

Table (3): Effect of long-term exposure of TCDD and TCDD + Vit. C on cholesterol, triglycerides, glucose and  thyroxine hormone (T4) levels of albino rats.

* Significant at P <0.05 to 0.01 in comparison with control.

 The letter a indicates significant differences between Vit. C- treated and non treated groups.

DISCUSSION:

                A significant decrease in both feed intake and body weight gain was recorded in long- term toxicity groups. These results were in agreement with Lutz et al. (1994), Lebofsky and Rozman,(1999) and Dunlap et al. (1999), they reported that TCDD in very low level doses causes highly decrease in body weight or wasting syndrome. Animal loss about 10% of body weight in one week after TCDD administration, there was a dose dependant decrease in feed consumption and body weight gain indicative to the wasting syndrome (Schiller et al., 2001). Wasting syndrome attributed to TCDD is responsible for the loss of fat and muscle, where TCDD-mediated decline in the adipose lipoprotein lipase activity which induce lipolysis in adipose cells reflected clinically with an increase serum cholesterol and triglycerides levels and decrease serum glucose and thyroxine levels (Hans et al., 1999). Decrease in feed intake was attributed to anorexia which developed as a typical features of TCDD -induced lethality by inducing feed refusal, body weight loss and exhaustion of energy stores (Matti et al., 1995). Restoration of normal value of weight and feed intake did not occur till the end of experimental time in both Vit. C treated and non treated group.

                Hyperlipoproteinemia associated with increased level of both cholesterol and triglycerides levels could be attributed to toxic effect of TCDD on lipid metabolism (Mitrou et al., 2001). Lipid metabolism changes presented as an increase in both cholesterol and triglycerides levels can be explained as a result to liver damage and bile duct lesions (Contarow and Trumper, 1962 and Breckinridge et al., 1978). Hyperlipoprotinemia may be also due to changes in thyroid activity, reflected as hypothyroidism (Breckinridge et al., 1978). TCDD has a severe toxic effect on the thyroid gland reflected clinically as decrease in serum thyroxin levels at all dose levels (Ivens et al., 1993 and Battershill, 1994). Restoration of normal values of both cholesterol and triglycerides levels did not occure until the end of the experimental time in all experimental groups except triglycerides level which restored at 144 h of TCDD administration in acute toxicity.

                Hyperlipoproteinemia is an obvious syndrome induced by TCDD in different experimental groups. This syndrome developed as a result to direct toxic effect of TCDD on liver, inducing liver damage, obstruction of bile duct and thyroid gland activity, which reflected clinically by changes in lipid metabolism and hypothyroidism. Restoration of normal levels did not occur till the end of the experiment time in all groups indicating no value of Vit. C treatment.

 Serum glucose levels showed a significant decrease in all groups. These results were in agreement with Gorski et al.(1990) and Weber and Stahl (1994) who reported that Hypoglycemia is a well documented effect in TCDD treated rats. Hypoglycemia was attributed to impaired gluconegenic pathway in combination with reduced feed intake (Matti et al., 1995). Lethal hypoglycemia was due to a reduction in the expression of the key enzyme of gluconegenesis, phosphoenol pyruvate carboxykinase (Seltzer, 1989), and the key enzyme of tryptophan metabolism (Weber and Stahl, 1994). Viluksela et al. (1999) added that lethality of TCDD associated with decreased of key gluconegenic enzymes in liver leads to decrease in liver glycogen concentrations. This seems to be secondary to the reduced feed intake.

 Restoration of glucose levels to normal values did not occur till the end of experimental time in all experimental groups. Hypoglycemia, recorded in different experimental groups was due to TCDD toxicity on the liver glycogenesis mechanism, and also to feed refusal. Restoration of normal values did not occur in all experimental groups till the end of the experimental time, indicating no value of Vit. C treatment on this compartment.

 The obtained results revealed a significant decrease in thyroxine level in the serum in all experimental groups. The same results were recorded by Weber et al. (1992),Weber and Stahl (1994), Lutz et al. (1994) and Nishimura et al. (1999). They reported that TCDD-induced lethality appeared as a decrease in serum level of thyroxine (T4). Hypothyroidism was contributed to TCDD-effect on the thyroid gland where TCDD treatment in rodents resulting in hypertrophy and hyperplasia of thyroid follicular cells and ultimately leads to thyroid adenoma and carcinoma (Barter and Klaassen, 1992). Hypothyroidism also could be attributed to TCDD-induced T4 glucouronidation by UDP-glucouronosyltransferase-1 and enhanced biliary excretion of glucouronide, which lead to decrease in T4 concentration (Hans et al., 1999).

 Restoration of normal value of thyroxin did not occur in both acute toxicity and long term groups. While restoration occurred in Vit.C treated group after 2 weeks of TCDD stoppage. As TCDD-induced hypothyroidism in different experimental groups, this toxic effect is variable between the acute and long-term toxicity and with or without Vit. C treatment. This indicates that TCDD toxicity is dose and time dependent. Hypothyroidism was attributed to thyroid gland affection and also due to TCDD-induce T4 glucuronidation which responsible for T4 catabolism or degradation. Restoration of normal values was variable between Vit. C treated and non treated group, which indicate some value of Vit. C in the treatment of the TCDD toxicity. Changes in the carbohydrates and lipid metabolism are very prominent symptoms of high doses of TCDD resulting mostly in the loss of fat and muscles. The sever form of body-weight loss are described as wasting syndrome. TCDD-mediated wasting syndrome through its effect on the lipolysis activity. This syndrome is correlated with increase the cholesterol and triglycerides in the serum. The values of feed intake and body weight gain did not restored till the end of experiment in both Vit. C treated and non treated group indicate that no value of Vit. C treatment on the TCDD toxicity.

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فقدان الوزن کنتيجة لتعرض الفئران البيضاء لمرکب

 2، 3، 7، 8 - رباعى کلورو ثنائى بنزو-بارا-دايوکسيـن

محمود عبد الناصر، عادل شحاته، إيمان الشرقاوي، ضيفي سالم

قسم الطب الشرعى والسموم - کلية الطب البيطرى - جامعة أسيوط

    يعد التلوث البيئي من أهم مشکلات عالمنا المعاصر. وتشکل مبيدات الآفات على اختلاف أنواعها واحداً من أهم وأخطر تلک المصادر وذلک لتأثيرها المباشر على  مکونات البيئة من هواء وماء وتربة وغذاء . وتکمن خطورة هذه المرکبات في خاصيتها التراکمية وزيادة ترکيزاتها في التربة وکذا أنسجة وأعضاء الحيوانات والطيور والأسماک المتعرضة لها .  وکذلک في أنسجة الإنسان المتعرض لهذه المرکبات مما يجعلها مصدراً متجدداً للتلوث ولفترات طويلة. ومع تزايد الاهتمام بدراسة الآثار السلبية المتعددة والمختلفة نتيجة استخدام هذه المرکبات فقد تم اختيار مرکب الديوکسين لهذه الدراسة . يمتلک هذا المرکب خصوصية شديدة لکونه لا يستخدم تجاريا أو صناعيا إلا أنه ينبعث کناتج جانبي أو فرعى عند تصنيع بعض المبيدات العشبية أو عند تعرضها لدرجات حرارة عالية مثل مرکب الـ   2,4-D مما يتسبب في انبعاث کميات کبيرة من  الديوکسين عند تصنيعه لعدم انضباط درجة الحرارة أو عند تعرضه لدرجات حرارة عالية بعد التصنيع. ولذلک تم اختيار مرکب التتراکلورو داى بنزوديوکسين (TCDD)  لهذه الدراسة لمعرفة بعض التأثيرات السمية له وتم اختيار فئران التجارب البيضاء کموديل (أو حيوانات تجارب) لإجراء هذه الدراسة وذلک لحساسيتها المتوسطة لهذا المرکب .

            ولقد تم استخدام عدد 200 فأر أبيض ذکر عمر 10 – 12 أسبوع تراوحت أوزانها عند التجربة بين100-150جرام و تم أقلمتها وتربيتها في أقفاص الفئران الخاصة بهذا الغرض. ويهدف البحث إلى دراسة التأثيرات السامة لمادة الديوکسين على الفئران البيضاء من حيث دراسة أثر الديوکسين على  معدل الزيادة في وزن الجسم واستهلاک الغذاء وکذلک على مستوى کل من الکوليسترول والتراى جليسريد بالإضافة إلي مستوى هرمون الثيروکسين وسکر الجلوکوز في مصل الدم . وکذلک دراسة تأثير فيتامين ج کمحاولة للعلاج في حالة التعرض طويل المدى لمادة الديوکسين. 

            تم تقسيم هذه الفئران إلى  خمس مجموعات بکل منها أربعون فارا. تم إعطاء المجموعة الأولى مرکب الديوکسين بواقع 4,.  ميکروجرام/کيلوجرام من وزن الجسم والمساوي لـ 1/5 الجرعة المتوسطة المميتة لهذا المرکب مذابة في زيت الذرة مرة واحدة فقط عن طريق الفم بأنبوب اللي المعدي بينما ترکت المجموعة الثانية ضابطة للتجربة الأولى . المجموعة الثالثة  تم إعطاءها الديوکسين بواقع 44,. ميکروجرام/کيلوجرام من وزن الجسم والمساوي لـ 1/50  من الجرعة  المميتة المتوسطة  مذابة في زيت الذرة يوماً بعد يوم ولمدة 12 أسبوع عن طريق الفم بأنبوبة اللي المعدي . أما المجموعة الرابعة فقد عوملت بنفس الجرعة السابقة مع إضافة فيتامين ج لماء الشرب بواقع 1 جم/لتر ماء. بينما ترکت المجموعة الخامسة والأخيرة کضابط للمجموعتين الثالثة والرابعة.

قد تم تجميع العينات بعد144,96,72,48,24,12 ساعة بعد التعرض لمرکب الديوکسين في المجموعتين الأولى والثانية  بينما تم تجميع العينات في المجموعات الثالثة والرابعة والخامسة بعد  4 و6 و8  و10  و12  أسبوع من التعرض, کما تم أيضا متابعة الفئران وأخذ العينات لمدة أسبوعين بعد إيقاف التعرض. وکذلک فقد تم وزن کمية الغذاء المستهلک وأيضا وزن الجسم في المجموعتين  الثالثة والرابعة ومقارنتهما بالمجموعة الضابطة   طوال فترة التجربة, وتم فصل مصل الدم واستخدامه في قياس ترکيز  بعض نواتج التمثيل الغذائي (الکوليسترول – التراىجليسريد والجلوکوز) وقياس مستوى هرمون الثيروکسين في الدم .

 أظهرت النتائج انخفاض معنوي في   کل من معدل الزيادة في وزن الجسم وأيضا في کمية الغذاء المستهلک مقارنة بالمجموعة الضابطة.کما سجلت النتائج نقص في مستوى الجلوکوز والثيروکسين في الدم وکذلک أظهرت النتائج زيادة معنوية في نسبة کل من الکوليسترول والدهون الثلاثية.

    وقد استخلص من نتائج هذه الدراسة أن التعرض  للديوکسين في کل من التسمم الحاد وطويل المدى يؤدى إلى  انخفاض ملحوظ في وزن الجسم وأيضا في کمية الغذاء المستهلک في الفئران المتعرضة, مصحوبا بانخفاض ملحوظ في مستوى کل من الجلوکوز وهرمون الثيروکسين في الجسم ومن هذا يتضح أن التغيرات التي تحدث في التمثيل الغذائي للمرکبات الدهنية والکربوهيدراتية هي من أهم النتائج التي تلي التعرض الطويل المدى للديوکسين مؤديا إلى فقد الدهون ونقص في وزن العضلات من الجسم
(wasting syndrome) ولم يظهر أي تأثير ملحوظ لفيتامين ج لعلاج هذه التغيرات الناتجة من التعرض لمرکب الديوکسين.   

                ولذلک ينصح  باستبدال المرکبات المستخدمة کمبيدات أو کمواد حافظة للأخشاب والتي ينبعث من تصنيعها أو استخدامها هذا المرکب المعروف باسم الديوکسين بأخرى تکون صديقة للبيئة . کما ينصح بعمل مسح دوري للتربة وللمنتجات الغذائية على اختلاف أنواعها لمرکب الديوکسين وخصوصاً المنتجات الغذائية المحتوية على المنتجات الحيوانية کاللحوم والألبان . کما ينصح  بتلافى المصادر الأخرى لهذا المرکب بمنع حرق المخلفات البلاستيکية والتخلص منها بطريقة علمية صحيحة لکي نمنع مصدراً رئيسياً لتلوث البيئة بهذا المرکب الخطير على الصحة العامة للإنسان والحيوان على حد سواء . کما تشير نتائج هذه الدراسة إلى أهمية إعادة البحث بصورة أوسع لبيان تأثير المرکبات المضادة للأکسدة عامة وفيتامين ج بصورة خاصة في الوقاية من الآثار السامة لهذا المرکب.