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Comparative toxicity studies in birds using nimesulide and diclofenac sodium
Environmental Toxicology and Pharmacology 22 (2006) 142–147
Comparative toxicity studies in birds using nimesulide and diclofenac sodium N.C. Prakash Reddy a,∗ , Y. Anjaneyulu b , B. Sivasankari a , K. Ananda Rao a a
b
Indian Immunologicals Limited, Rakshapuram, Gachibowli, Hyderabad 500 019, India Department of Veterinary Pathology, Veterinary College, ANGRAU, Rajendranagar, Hyderabad, India Received 4 June 2005; accepted 4 February 2006 Available online 29 March 2006
Abstract Nimesulide, a sulfonanilide derivative, was compared with diclofenac sodium for toxicity in poultry. In this study, Vanaraja and PB1 birds of 6 weeks old (either sex) were mixed and equally divided into 5 groups of 10 birds each. The birds were inoculated with nimesulide, @ 5 and 2 mg; vehicle @ 0.5 ml; and diclofenac sodium @ 5 mg on kg bwt basis. One group served as untreated control. All the groups were observed for a period of 28 days. Forty percent mortality was observed within 12 days in diclofenac-treated group. While birds inoculated with nimesulide remained normal. No significant differences in the weight gain, haematology, total protein contents in the nimesulide and diclofenac groups (survived birds) were observed when compared with the control group of birds. Serum creatinine, cholesterol, alkaline phosphatase, and aspartate aminotransferase levels were significantly (P < 0.05) high in diclofenac-treated group compared to nimesulide (P > 0.05) and control groups. Nimesulide-treated groups did not show any histopathological lesions, where as diclofenac-treated birds showed histopathological lesions in liver and kidney. © 2006 Elsevier B.V. All rights reserved. Keywords: Nimesulide; Diclofenac; Haematology; Seum biochemistry; Histopathology
1. Introduction Diclofenac sodium is extensively used in the treatment of animals in Indian subcontinent, Pakistan and Bangladesh. While the drug is being extensively used in the treatment of animals, it lead to the mortality of three vulture species in India and Pakistan mainly due to diclofenac-induced visceral gout. The widespread mortality and decline of three vulture species (Gyps bengalensis, Gyps indicus and Gyps tenuirostris) in southern Asia has been a cause of concern since widely reported in 1999 (Ali and Ripley, 1968; Pain, 2003). Following observed declines in western India, there were reports of further vulture mortalities in Pakistan (Gilbert, 2002). High levels of diclofenac residues in the tissues of dead vultures were attributed to scavenging of dead domesticated animals previously inoculated with diclofenac by vultures led to the conclusion of possible involvement of diclofenac for death of vultures and consequent decline ∗
Corresponding author. Tel.: +91 40 23000211/212; fax: +91 40 2300213. E-mail addresses: [email protected], [email protected] (N.C. Prakash Reddy). 1382-6689/$ – see front matter © 2006 Elsevier B.V. All rights reserved. doi:10.1016/j.etap.2006.02.004
in their population. Later, diclofenac residues and renal disease (visceral gout) were experimentally reproduced in Oriental white-backed vultures (Lindsay Oaks and Martin, 2004). Keeping in view of vulture mortalities, the government of India is planning to ban the use of diclofenac sodium usage in animals. In the present circumstances, the other alternative drug currently available in India is nimesulide. Hence, the present study was taken up to compare and assess the subacute toxicity of nimesulide and dicofenac in birds. Nimesulide, a potent non-steroidal anti-inflammatory drug (NSAID) belongs to sulfonanilide derivatives, exerts its effect by inhibiting Cyclooxygenase-2 (COX-2) (Garcia-Nieto et al., 1999; Shah et al., 1999), which is inducible by cytokines and other inflammatory mediators during inflammation without affecting COX-1 activity in gastroduodenal mucosa even at maximal therapeutic dose when compared to diclofenac, naproxen or ibuprofen (Tripati, 2003). The drug also exerts its effects by other mechanisms like reduced generation of superoxide by neutrophils, inhibition of platelet activation factor synthesis (PAF) and tumor necrosis factor release (TNF␣), free radical scavenging and inhibition of metalloproteinase activity in cartilage.
N.C. Prakash Reddy et al. / Environmental Toxicology and Pharmacology 22 (2006) 142–147
Diclofenac sodium, a non-steroidal anti-inflammatory drug belongs to the arylacetic acid derivative, exerts its antiinflammatory, analgesic and antipyretic action by inhibiting both inflammatory and physiological prostaglandin synthesis. Physiological prostaglandins are required for maintenance of normal gastric mucosa. Hence, the adverse effects are mainly gastric pain, gastric irritation, gastric mucosal bleeding followed by ulcerations. The drug also has the property of reversible elevation of serum aminotransferase, which in turn result in kidney and liver damage. The efficacy and safety of nimesulide (injectable) was well established and studied in different animal models in our earlier studies (data not published). But the data on safety of nimesulide in birds is currently lacking. This report describes the sub acute toxicity studies using nimesulide and diclofenac sodium injections in Vanaraja (Synthetic female × Red Cornish) and PB1 poultry breeds.
(b)
(c)
2. Materials and methods 2.1. Birds Poultry breeds: (a) Vanaraja (6 weeks old, of either sex) (b) PB1 (6 weeks old, of either sex) All the groups were kept under standard conditions and fed with commercially available feed with potable water.
2.2. Drugs (a) Nimesulide (Nimovet® ) (10% veterinary injectable nimesulide preparation; Indian Immunologicals Limited, Hyderabad), (b) Diclofenac sodium (Diclodin® ) (injectable diclofenac sodium 25mg/ml; Indian Immunologicals Limited, Hyderabad), (c) Vehicle (propylene glycol, used in the Nimovet® injection).
(d)
2.3. Diagnostic kits (Qualigens; Glaxo) (e) (a) (b) (c) (d) (e)
Creatinine Cholesterol Total protein Alkaline phosphatase (ALP) Aspartate aminotrasferase (AST)
2.4. Methods (a) Vanaraja and PB1 birds of either sex were randomly divided into 5 groups of 10 birds each and inoculated once intramuscularly with various drugs as follows: (1) Group-I: Nimovet injection @ 5 mg/kg bwt. This is the upper dosage limit recommended by the manufacturer. (2) Group-II: Nimovet injection @ 2 mg/kg bwt. This particular dosage was chosen based on the recommendation of the manufacturers, which ranges from 2–5 mg/kg bwt depending on the severity of the case. (3) Group-III: Vehicle alone (propylene glycol, used in Nimovet injection) @ 0.5 ml/kg bwt. (4) Group-IV: Diclodin sodium injection @ 5 mg/kg bwt. Here, diclofenac is dissolved in distilled water and hence vehicle control is not included to minimize the pain and unwanted use of birds in the experimentation. Here, the dose given was higher to simulate the field conditions where the animal receives abnormal doses by paraveterinarians and animal
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owners in Indian subcontinent. Others have already studied the normal doses (2.5 mg/kg bwt), but the number of birds used in those studies was too small to conclude. (5) Group-V: Control birds without medication. Blood samples: Blood samples were collected on weekly intervals till 4 weeks from wing vein of five birds randomly selected from each group into a set of sterilized glass tubes containing disodium ethylene diamine tetra acetic acid (Na2 EDTA) for determination of haematological parameters and second set of glass tubes without anticoagulant for serum separation. Sera samples were stored at –20 ◦ C till further use. Analytical procedures: The weight gain of the birds was assessed by weighing the birds daily for 28 days. The packed cell volume (PCV), total erythrocyte count (TEC) and total leukocyte count (TLC) was determined using standard methods. Biochemical parameters like, total serum protein, serum cholesterol, serum creatinine, alkaline phosphatase (ALP) and aspartate amino trasnferase (AST) were estimated using standard diagnostic kits according to manufacturer (Qualigens, Glaxo) instructions. Total serum proteins: Liver except immunoglobulins produces almost all proteins in the serum. The total serum protein estimation serves as an important tool for assessing the functioning of liver, malabsorption from intestine due to inflammations, malnutrition, glomerulonephropathy, acute inflammations, etc. Cholesterol: Serum cholesterol levels are good indication of tissue and hepatocyte metabolism, pancreatic malfunctions, and nephrotic syndrome. Serum creatinine: Most of the creatinine originates from the nonenzymatic conversion of creatinine in muscle. The creatinine thus produced is filtered by glomerular filtration. Altered levels of creatinine along with AST are good indicators of muscle and liver damage. Alkaline phosphatase: Alkaline phosphatase (ALP) is present in liver, bound to the cells. This enzyme is also present to some extent, in all tissues but only few organs contribute to the circulating enzyme level. This enzyme plays a role in detoxification of endotoxins in the liver mainly. The altered levels of this enzyme are observed in active liver damage, extra-hepatic and intra-hepatic biliary obstructions. Aspartate aminotransferase (AST): This enzyme is present in many tissues and is useful in evaluating muscle and liver damage. Specimen collection: The treatment and control group birds were sacrificed on 28th day post-treatment. The vital organs namely heart, liver, spleen, kidney, lungs and intestines were collected in 10% formalin until they are processed for histopathology. The sections of the organs were stained using HE (hematoxilin and eosin) and viewed under 160× magnifying lens. The results were analyzed based on the comparative values of the treatment groups and control group. Statistical analysis: Statistical analysis was done to test of significance between control and treatment groups using t-test by methods described by Snedecor and Cochran (1967).
3. Results All the nimesulide-treated groups of birds including vehicletreated group remained healthy during post-treatment period of 28 days, whereas diclofenac treatment group showed 40% mortality within 12 days. Weight gain of birds in each group was recorded daily using a calibrated digital balance in kilograms. No significant difference was observed in weight gain of birds between the treatment groups and control group. Haematological parameters like Hb%, PCV%, TEC (cu.mm) and TLC (103 /cu.mm) were estimated on weekly basis. The results indicate no significant change in the values of Hb, PCV, TEC and TLC of treatment and control groups (Table 1). The 28th day serum samples were also tested for biochemical parameters like total proteins, serum-creatinine, serum-cholesterol, and serum-alkaline phosphatase and serum
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Table 1 Results of weight gain, haematological and biochemical parameters Parameters
Group-I: Nimovet @ 5 mg
Weight gain Hb (%) PCV (%) TEC (cu.mm) TLC (103 /cu.mm) Total protein (g%) Creatinine (mg%) Cholesterol (mg%) ALP (KA units) AST (IU/l)
1.9 10.49 30 3.70 18.65 4.63 1.71 109.81 15.27 31
± ± ± ± ± ± ± ± ± ±
0.233 1.32 1.74 0.44 1.25 0.2 0.2 6.07 2.0 1.01
Group-II: Nimovet @ 2 mg 1.73 10.21 29.11 3.99 18.708 4.96 1.73 112.67 15.95 32
± ± ± ± ± ± ± ± ± ±
0.18 1.5 2.34 0.27 1.72 .0.46 0.22 3.62 1.78 1.1
Group-III: vehicle (propylene glycol) 1.82 10 30.1 4.12 18.41 4.72 1.69 116.67 16.23 31
± ± ± ± ± ± ± ± ± ±
0.32 0.98 2.29 0.40 1.56 0.25 0.16 4.0 0.82 0.8
Gropu-IV: diclodin @ 5 mg 1.87 10.81 28.23 4.00 18.98 4.53 1.91 134.63 33.68 39.66
± ± ± ± ± ± ± ± ± ±
0.22 1.17 2.29 0.38 1.41 0.31 0.16* 3.33* 3.24* 1.81*
Group-V: control 1.89 10.08 26.6 4.16 18.69 4.18 1.70 113.01 15.00 31
± ± ± ± ± ± ± ± ± ±
0.31 1.3 2.36 0.39 1.43 1.43 0.25 5.94 1.03 1.66
Values are mean ± S.D. of observations. * The values indicate significance. P < 0.05 (t-test) in relation to control.
aspartate aminotransferase. Creatinine results showed significantly (P < 0.05) elevated levels (1.917 ± 0.16) in group-IV (diclofenac) on 28th day when compared to that of other treatment groups and control group (1.7 ± 0.25). The serum cholesterol results clearly showed significantly (P < 0.05) elevated level (134.63 ± 3.33) when compared to treatment and control (113.01 ± 5.9) group. Serum alkaline phosphatase and aspartate transaminase estimates in diclofenac sodium-treated group (groupIV) showed significantly high values (33.68 ± 3.24; 39.66 ± 1.81; P < 0.05) when compared to that of other treatment groups and control group (15 ± 1.03; 31 ± 1.66) (Table 1). The histopathological sections were made from the vital organs (kidney, liver, heart, spleen, intestine and lungs) collected on the 28th day indicated kidney and liver damage in group-IV treated with diclofenac sodium. The kidney sections showed mild tubular degeneration with lymphoid aggregates (Fig. 1); disruption of tubular architecture with mild inter-tubular fibrosis (Fig. 2) and marked inter-tubular congestion (Fig. 3) when compared to the healthy kidney sections of control group (Fig. 4) and nimesulide treatment group (Fig. 4a). The liver sections in diclofenac-treated group showed severe sinusoidal and central vein congestion (Fig. 5); marked central vein and sinusoidal congestion with bile duct hyperplasia (Fig. 6) when compared to the healthy kidney sections of control group (Fig. 7) and nimesulide treatment group (Fig. 7a). However, the
Fig. 2. Kidney. Disrupted tubular architecture with mild inter-tubular fibrosis (HE× 160).
Fig. 3. Kidney. Marked inter-tubular congestion (HE× 160).
organs like heart, spleen and lungs did not show any pathological lesions. 4. Discussion
Fig. 1. Kidney. Mild tubular degeneration with lymphoid aggregates (HE× 160).
Nimesulide, propylene glycol (vehicle control) and control groups remained healthy and normal during post-treatment
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Fig. 4. Kidney. Normal kidney section (HE× 160). (a) Kidney section of nimesulide-treated group (HE× 160).
Fig. 5. Liver. Severe sinusoidal congestion and central vein congestion (HE× 160).
Fig. 6. Liver. Marked central vein and sinusoidal congestion with bile duct hyperplasia (HE× 160).
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Fig. 7. Liver. Normal liver section (HE× 160). (a) Liver section nimesulidetreated group (HE× 160).
period of 28 days. But, in diclofenac-treated group, 40% acute deaths were observed leaving us no time to investigate any of the parameters we adopted in the current study. This is in agreement with the observations made in Punjab province of Pakistan, where vultures were healthy and in good condition with good body fat before they were found dead even when they were incubating the eggs (Riley et al., 2003) due to visceral gout. But no such observations of gout noticed in the present study. In this study, though we have used higher dose of the drug keeping in view the abnormal dose administered to the live stock in India by paraveterinarians and live stock owners, did not produce 100% mortality, which is, contradictory to the observations made by others (Lindsay Oaks and Martin, 2004) who used 2.5 mg/kg bwt which resulted in 100% mortality of vultures due to nephrotoxicity. We are of the opinion that those investigators have used only two vultures and that is not statistically significant number always. Our results indicated that, domestic fowl might be partially resistant to diclofenac compared to scavenging birds like vultures. Weight gain, haemoglobin, total leukocyte count, total erythrocyte count, packed cell volume (PCV) and total serum proteins have shown no significant changes in the values in any of the treatment, propylene glycol and control groups compared to the normal values reported for birds (Jain, 1993). Some researchers reported significant decrease in TLC values in Japanese quails when treated with furazolidone (Coles, 1986), but nimesulide, diclofenac and propylene glycol did not cause decrease in TLC in the present study.
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Creatinine and AST levels were significantly high in diclofenac group compared to the treatment and control groups indicating the probable muscular and hepatocellular damage caused by diclofenac. It is observed that the levels of creatinine and AST shoots up in case of hepatocellular and muscular damage (Lumeij, 1997; Lumeij and Waterhof, 1987), both values forms a good tool for assessing liver and muscle damage (Dabbert and Powell, 1993). In the present study, AST levels in the diclofenac group remained significantly elevated at the end of 28 days of experimentation. Our observations are in contradictory to the observations made by others (Kendel and Harr, 2002) where they observed elevated levels of AST for 100 h following doxycycline treatment and later dropped down to normal levels. Increased levels of AST were also noticed due to hepatocellular degenerative changes caused by furazolidone in Japanese quails (Nazifi and Asasi, 2001; Arbid et al., 1990). Serum cholesterol levels were found to be in normal range during the post-experimentation period of 28 days in nimesulide, vehicle control and control groups. Diclofenactreated group showed elevated levels of cholesterol probably indicating the renal damage (Coles, 1986). Elevated levels of cholesterol are usually observed in birds during vitellogenesis and egg formation (Johnson, 2000). But in the present study, abnormal serum cholesterol levels could be mainly due to renal damage but not due to vitellogenesis or egg formation, as all the birds used in the experimentation were of 6 weeks age. Alkaline phosphatase levels remained in normal levels as reported in some of avian species (Kendel and Harr, 2002; Simaraks et al., 2004) in all the treatment and control groups except diclofenac group. Elevated levels of ALP in diclofenac group are indicative of liver damage (Lumeij and Waterhof, 1987). The vehicle (propylene glycol,) control group did not show significant elevation of any of the parameters tested, which is in agreement with others who noticed that PG is non toxic to chicken (Hill and Camardese, 1981). All the above observations were supported by the altered architecture of kidney and liver sections upon histopathological examination after 28 days of post-treatment. However, we are not presenting all the histopathological data regarding spleen, heart, intestine and lungs, as there were no lesions observed in those organs. Kidney sections in diclofenac group showed, disruption of tubular architecture, lymphoid aggregation, intertubular fibrosis and marked congestion which is in agreement with, tubular necrosis and fibrosis observed in Oriental whitebacked vultures due to diclofenac poisoning (Lindsay Oaks and Martin, 2004). NSAIDs, including diclofenac, are also recognized as causing renal disease in mammals (Murray and Brater, 1993; Todd and Sorkin, 1998). Liver sections in diclofenac group showed, severe sinusoidal congestion with bileduct hyperplasia indicating liver injury caused by diclofenac sodium injection. To our knowledge, there was no published information available about adverse effects of nimesulide on haemopoetic system, liver, kidney muscle, intestine and spleen in avians. Hence, it was concluded that nimesulide and the vehicle (propylene gly-
col) used in the nimesulide injection preparation are completely safe for birds at the dose levels tested. Acknowledgements We extend our sincere gratitude to Dr. V.A. Srinivasan, Executive Director, Indian Immunologicals Limited, Hyderabad for his expert guidance in preparing the manuscript. We also thank Dr. B. Shoba, University of Hyderabad, Hyderabad for the help rendered to carry out the statistical analysis of the data. References Ali, S., Ripley, S.D., 1968. Handbook of the Birds of India and Pakistan Together with Those of Nepal, Bhutan, and Ceylon. I. Divers and Hawks. Oxford University Press, Oxford. Arbid, M.S., EL-Habbak, M.M.E., Hanafy, M.S.M., 1990. Toxicological and biological studies on Japanese quails fed graded levels of furazolidone. Acta. Vet. Hung. 38, 271–279. Coles, E.H., 1986. Veterinary Clinical Pathology, fourth ed. W.B. Saunders Co., Philadelphia. Dabbert, C.B., Powell, K.C., 1993. Serum enzyme as indicators of capture myopathy in mallards (Anas platyrhynchos). J. Wildlife Dis. 29 (2), 304–309. Garcia-Nieto, R., Perez, C., Checa, A., Gago, F., 1999. Molecular model of the interaction between nimesulide and human cuclooxygenase-2. Rheumatology 38 (Suppl. 1), 14–18. Gilbert, M., 2002. Breeding and mortality of Oriental white-backed vulture Gyps bengalensis in Punjab Province Pakistan. Bird Conserv. Int. 12, 311–326. Hill, E.F., Camardese, M.B., 1981. In: Lamb, D.W., Kenega, E.E. (eds.), Avian and Mammalian Wildlife. Toxicology: Second Conference, ASTM STP 757. American Society for Testing Material, pp. 41– 65. Johnson, A.L., 2000. Reproduction in female. In: Whittow, G.C. (Ed.), Sturkies Avian Physiology. Academic Press, San Diego, Calif., pp. 569–596. Jain, N.C., 1993. Essentials of Veterinary Haematology. Lea and Febiger, Philadelphia. Kendel, E., Harr, 2002. Clinical chemistry of companion avian species: a review. Vet. Clin. Pathol. 31 (3), 140–151. Lumeij, J.T., 1997. Avian clinical biochemistry. In: Kaneko, J.J., Harvey, J.W., Bruss, M.L. (Eds.), Clinical Biochemistry of Domestic Animals. Academic Press, San Diego, Calif., pp. 857–884. Lumeij, J.T., Waterhof, I., 1987. Blood chemistry for the diagnosis of hepatocellular disease in birds: a review. Vet. Q. 9, 255– 267. Lindsay Oaks, J., Martin, G., 2004. Diclofenac residues as the cause of vulture population decline in Pakistan. Nature, 1–4, Letters. Murray, M.D., Brater, D.C., 1993. Renal toxicity of non-steroidal anti-inflammatory drugs. Annu. Rev. Pharmacol. Toxicol 33, 435– 465. Nazifi, S., Asasi, 2001. Haematological and serum biochemical studies on Japanese quails (Coturnix coturnix japanica) fed different levels of furazolidone. Rev. Med. Vet. 152 (10), 705–708. Pain, D., 2003. Causes and effects of temperospatial declines of Gyps vultures in Asia. Conserv. Biol. 17, 661–671. Riley, J., Oaks, J.L., Gilbert, M., 2003. Raillietiella trachea n.sp., a pentastomid from the trachea of an Oriental white backed vulture (Gyps bengalensis) taken in Pakistan, with speculation about its life cycle. Syst. Parasitol. 56 (2), 155–561. Simaraks, S., Chinrasri, O., Aengwanich, S., 2004. Haematological, electrolyte and serum biochemical values of the Thai indigenous chickens
N.C. Prakash Reddy et al. / Environmental Toxicology and Pharmacology 22 (2006) 142–147 (Gallus domesticus) in norteastern Thailand. Songklanakarin J. Sci. Technol. 26 (3), 425–430. Shah, A.A., Murray, F.E., Fitzgerald, D.J., 1999. The in vivo assessment of nimesulide COX-2 selectivity. Rheumatology 38 (Suppl. 1), 19–23. Snedecor, G.W., Cochran, W.G., 1967. Statistical Methods, sixth ed. Oxford and I.B.H Publishing Co. Pvt. Ltd., New Delhi.
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Todd, P.A., Sorkin, E.M., 1998. Diclofenac sodium, a reappraisal of its pharmacokinetic and pharmacodynamic properties, and therapeutic efficacy. Drugs 35, 244–285. Tripati, K.D., 2003. Essentials of Medical Pharmacology, fourth ed. Jaypee Brothers, New Delhi, pp. 462–464.
Comparative toxicity studies in birds using nimesulide and diclofenac sodium N.C. Prakash Reddy a,∗ , Y. Anjaneyulu b , B. Sivasankari a , K. Ananda Rao a a
b
Indian Immunologicals Limited, Rakshapuram, Gachibowli, Hyderabad 500 019, India Department of Veterinary Pathology, Veterinary College, ANGRAU, Rajendranagar, Hyderabad, India Received 4 June 2005; accepted 4 February 2006 Available online 29 March 2006
Abstract Nimesulide, a sulfonanilide derivative, was compared with diclofenac sodium for toxicity in poultry. In this study, Vanaraja and PB1 birds of 6 weeks old (either sex) were mixed and equally divided into 5 groups of 10 birds each. The birds were inoculated with nimesulide, @ 5 and 2 mg; vehicle @ 0.5 ml; and diclofenac sodium @ 5 mg on kg bwt basis. One group served as untreated control. All the groups were observed for a period of 28 days. Forty percent mortality was observed within 12 days in diclofenac-treated group. While birds inoculated with nimesulide remained normal. No significant differences in the weight gain, haematology, total protein contents in the nimesulide and diclofenac groups (survived birds) were observed when compared with the control group of birds. Serum creatinine, cholesterol, alkaline phosphatase, and aspartate aminotransferase levels were significantly (P < 0.05) high in diclofenac-treated group compared to nimesulide (P > 0.05) and control groups. Nimesulide-treated groups did not show any histopathological lesions, where as diclofenac-treated birds showed histopathological lesions in liver and kidney. © 2006 Elsevier B.V. All rights reserved. Keywords: Nimesulide; Diclofenac; Haematology; Seum biochemistry; Histopathology
1. Introduction Diclofenac sodium is extensively used in the treatment of animals in Indian subcontinent, Pakistan and Bangladesh. While the drug is being extensively used in the treatment of animals, it lead to the mortality of three vulture species in India and Pakistan mainly due to diclofenac-induced visceral gout. The widespread mortality and decline of three vulture species (Gyps bengalensis, Gyps indicus and Gyps tenuirostris) in southern Asia has been a cause of concern since widely reported in 1999 (Ali and Ripley, 1968; Pain, 2003). Following observed declines in western India, there were reports of further vulture mortalities in Pakistan (Gilbert, 2002). High levels of diclofenac residues in the tissues of dead vultures were attributed to scavenging of dead domesticated animals previously inoculated with diclofenac by vultures led to the conclusion of possible involvement of diclofenac for death of vultures and consequent decline ∗
Corresponding author. Tel.: +91 40 23000211/212; fax: +91 40 2300213. E-mail addresses: [email protected], [email protected] (N.C. Prakash Reddy). 1382-6689/$ – see front matter © 2006 Elsevier B.V. All rights reserved. doi:10.1016/j.etap.2006.02.004
in their population. Later, diclofenac residues and renal disease (visceral gout) were experimentally reproduced in Oriental white-backed vultures (Lindsay Oaks and Martin, 2004). Keeping in view of vulture mortalities, the government of India is planning to ban the use of diclofenac sodium usage in animals. In the present circumstances, the other alternative drug currently available in India is nimesulide. Hence, the present study was taken up to compare and assess the subacute toxicity of nimesulide and dicofenac in birds. Nimesulide, a potent non-steroidal anti-inflammatory drug (NSAID) belongs to sulfonanilide derivatives, exerts its effect by inhibiting Cyclooxygenase-2 (COX-2) (Garcia-Nieto et al., 1999; Shah et al., 1999), which is inducible by cytokines and other inflammatory mediators during inflammation without affecting COX-1 activity in gastroduodenal mucosa even at maximal therapeutic dose when compared to diclofenac, naproxen or ibuprofen (Tripati, 2003). The drug also exerts its effects by other mechanisms like reduced generation of superoxide by neutrophils, inhibition of platelet activation factor synthesis (PAF) and tumor necrosis factor release (TNF␣), free radical scavenging and inhibition of metalloproteinase activity in cartilage.
N.C. Prakash Reddy et al. / Environmental Toxicology and Pharmacology 22 (2006) 142–147
Diclofenac sodium, a non-steroidal anti-inflammatory drug belongs to the arylacetic acid derivative, exerts its antiinflammatory, analgesic and antipyretic action by inhibiting both inflammatory and physiological prostaglandin synthesis. Physiological prostaglandins are required for maintenance of normal gastric mucosa. Hence, the adverse effects are mainly gastric pain, gastric irritation, gastric mucosal bleeding followed by ulcerations. The drug also has the property of reversible elevation of serum aminotransferase, which in turn result in kidney and liver damage. The efficacy and safety of nimesulide (injectable) was well established and studied in different animal models in our earlier studies (data not published). But the data on safety of nimesulide in birds is currently lacking. This report describes the sub acute toxicity studies using nimesulide and diclofenac sodium injections in Vanaraja (Synthetic female × Red Cornish) and PB1 poultry breeds.
(b)
(c)
2. Materials and methods 2.1. Birds Poultry breeds: (a) Vanaraja (6 weeks old, of either sex) (b) PB1 (6 weeks old, of either sex) All the groups were kept under standard conditions and fed with commercially available feed with potable water.
2.2. Drugs (a) Nimesulide (Nimovet® ) (10% veterinary injectable nimesulide preparation; Indian Immunologicals Limited, Hyderabad), (b) Diclofenac sodium (Diclodin® ) (injectable diclofenac sodium 25mg/ml; Indian Immunologicals Limited, Hyderabad), (c) Vehicle (propylene glycol, used in the Nimovet® injection).
(d)
2.3. Diagnostic kits (Qualigens; Glaxo) (e) (a) (b) (c) (d) (e)
Creatinine Cholesterol Total protein Alkaline phosphatase (ALP) Aspartate aminotrasferase (AST)
2.4. Methods (a) Vanaraja and PB1 birds of either sex were randomly divided into 5 groups of 10 birds each and inoculated once intramuscularly with various drugs as follows: (1) Group-I: Nimovet injection @ 5 mg/kg bwt. This is the upper dosage limit recommended by the manufacturer. (2) Group-II: Nimovet injection @ 2 mg/kg bwt. This particular dosage was chosen based on the recommendation of the manufacturers, which ranges from 2–5 mg/kg bwt depending on the severity of the case. (3) Group-III: Vehicle alone (propylene glycol, used in Nimovet injection) @ 0.5 ml/kg bwt. (4) Group-IV: Diclodin sodium injection @ 5 mg/kg bwt. Here, diclofenac is dissolved in distilled water and hence vehicle control is not included to minimize the pain and unwanted use of birds in the experimentation. Here, the dose given was higher to simulate the field conditions where the animal receives abnormal doses by paraveterinarians and animal
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owners in Indian subcontinent. Others have already studied the normal doses (2.5 mg/kg bwt), but the number of birds used in those studies was too small to conclude. (5) Group-V: Control birds without medication. Blood samples: Blood samples were collected on weekly intervals till 4 weeks from wing vein of five birds randomly selected from each group into a set of sterilized glass tubes containing disodium ethylene diamine tetra acetic acid (Na2 EDTA) for determination of haematological parameters and second set of glass tubes without anticoagulant for serum separation. Sera samples were stored at –20 ◦ C till further use. Analytical procedures: The weight gain of the birds was assessed by weighing the birds daily for 28 days. The packed cell volume (PCV), total erythrocyte count (TEC) and total leukocyte count (TLC) was determined using standard methods. Biochemical parameters like, total serum protein, serum cholesterol, serum creatinine, alkaline phosphatase (ALP) and aspartate amino trasnferase (AST) were estimated using standard diagnostic kits according to manufacturer (Qualigens, Glaxo) instructions. Total serum proteins: Liver except immunoglobulins produces almost all proteins in the serum. The total serum protein estimation serves as an important tool for assessing the functioning of liver, malabsorption from intestine due to inflammations, malnutrition, glomerulonephropathy, acute inflammations, etc. Cholesterol: Serum cholesterol levels are good indication of tissue and hepatocyte metabolism, pancreatic malfunctions, and nephrotic syndrome. Serum creatinine: Most of the creatinine originates from the nonenzymatic conversion of creatinine in muscle. The creatinine thus produced is filtered by glomerular filtration. Altered levels of creatinine along with AST are good indicators of muscle and liver damage. Alkaline phosphatase: Alkaline phosphatase (ALP) is present in liver, bound to the cells. This enzyme is also present to some extent, in all tissues but only few organs contribute to the circulating enzyme level. This enzyme plays a role in detoxification of endotoxins in the liver mainly. The altered levels of this enzyme are observed in active liver damage, extra-hepatic and intra-hepatic biliary obstructions. Aspartate aminotransferase (AST): This enzyme is present in many tissues and is useful in evaluating muscle and liver damage. Specimen collection: The treatment and control group birds were sacrificed on 28th day post-treatment. The vital organs namely heart, liver, spleen, kidney, lungs and intestines were collected in 10% formalin until they are processed for histopathology. The sections of the organs were stained using HE (hematoxilin and eosin) and viewed under 160× magnifying lens. The results were analyzed based on the comparative values of the treatment groups and control group. Statistical analysis: Statistical analysis was done to test of significance between control and treatment groups using t-test by methods described by Snedecor and Cochran (1967).
3. Results All the nimesulide-treated groups of birds including vehicletreated group remained healthy during post-treatment period of 28 days, whereas diclofenac treatment group showed 40% mortality within 12 days. Weight gain of birds in each group was recorded daily using a calibrated digital balance in kilograms. No significant difference was observed in weight gain of birds between the treatment groups and control group. Haematological parameters like Hb%, PCV%, TEC (cu.mm) and TLC (103 /cu.mm) were estimated on weekly basis. The results indicate no significant change in the values of Hb, PCV, TEC and TLC of treatment and control groups (Table 1). The 28th day serum samples were also tested for biochemical parameters like total proteins, serum-creatinine, serum-cholesterol, and serum-alkaline phosphatase and serum
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Table 1 Results of weight gain, haematological and biochemical parameters Parameters
Group-I: Nimovet @ 5 mg
Weight gain Hb (%) PCV (%) TEC (cu.mm) TLC (103 /cu.mm) Total protein (g%) Creatinine (mg%) Cholesterol (mg%) ALP (KA units) AST (IU/l)
1.9 10.49 30 3.70 18.65 4.63 1.71 109.81 15.27 31
± ± ± ± ± ± ± ± ± ±
0.233 1.32 1.74 0.44 1.25 0.2 0.2 6.07 2.0 1.01
Group-II: Nimovet @ 2 mg 1.73 10.21 29.11 3.99 18.708 4.96 1.73 112.67 15.95 32
± ± ± ± ± ± ± ± ± ±
0.18 1.5 2.34 0.27 1.72 .0.46 0.22 3.62 1.78 1.1
Group-III: vehicle (propylene glycol) 1.82 10 30.1 4.12 18.41 4.72 1.69 116.67 16.23 31
± ± ± ± ± ± ± ± ± ±
0.32 0.98 2.29 0.40 1.56 0.25 0.16 4.0 0.82 0.8
Gropu-IV: diclodin @ 5 mg 1.87 10.81 28.23 4.00 18.98 4.53 1.91 134.63 33.68 39.66
± ± ± ± ± ± ± ± ± ±
0.22 1.17 2.29 0.38 1.41 0.31 0.16* 3.33* 3.24* 1.81*
Group-V: control 1.89 10.08 26.6 4.16 18.69 4.18 1.70 113.01 15.00 31
± ± ± ± ± ± ± ± ± ±
0.31 1.3 2.36 0.39 1.43 1.43 0.25 5.94 1.03 1.66
Values are mean ± S.D. of observations. * The values indicate significance. P < 0.05 (t-test) in relation to control.
aspartate aminotransferase. Creatinine results showed significantly (P < 0.05) elevated levels (1.917 ± 0.16) in group-IV (diclofenac) on 28th day when compared to that of other treatment groups and control group (1.7 ± 0.25). The serum cholesterol results clearly showed significantly (P < 0.05) elevated level (134.63 ± 3.33) when compared to treatment and control (113.01 ± 5.9) group. Serum alkaline phosphatase and aspartate transaminase estimates in diclofenac sodium-treated group (groupIV) showed significantly high values (33.68 ± 3.24; 39.66 ± 1.81; P < 0.05) when compared to that of other treatment groups and control group (15 ± 1.03; 31 ± 1.66) (Table 1). The histopathological sections were made from the vital organs (kidney, liver, heart, spleen, intestine and lungs) collected on the 28th day indicated kidney and liver damage in group-IV treated with diclofenac sodium. The kidney sections showed mild tubular degeneration with lymphoid aggregates (Fig. 1); disruption of tubular architecture with mild inter-tubular fibrosis (Fig. 2) and marked inter-tubular congestion (Fig. 3) when compared to the healthy kidney sections of control group (Fig. 4) and nimesulide treatment group (Fig. 4a). The liver sections in diclofenac-treated group showed severe sinusoidal and central vein congestion (Fig. 5); marked central vein and sinusoidal congestion with bile duct hyperplasia (Fig. 6) when compared to the healthy kidney sections of control group (Fig. 7) and nimesulide treatment group (Fig. 7a). However, the
Fig. 2. Kidney. Disrupted tubular architecture with mild inter-tubular fibrosis (HE× 160).
Fig. 3. Kidney. Marked inter-tubular congestion (HE× 160).
organs like heart, spleen and lungs did not show any pathological lesions. 4. Discussion
Fig. 1. Kidney. Mild tubular degeneration with lymphoid aggregates (HE× 160).
Nimesulide, propylene glycol (vehicle control) and control groups remained healthy and normal during post-treatment
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Fig. 4. Kidney. Normal kidney section (HE× 160). (a) Kidney section of nimesulide-treated group (HE× 160).
Fig. 5. Liver. Severe sinusoidal congestion and central vein congestion (HE× 160).
Fig. 6. Liver. Marked central vein and sinusoidal congestion with bile duct hyperplasia (HE× 160).
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Fig. 7. Liver. Normal liver section (HE× 160). (a) Liver section nimesulidetreated group (HE× 160).
period of 28 days. But, in diclofenac-treated group, 40% acute deaths were observed leaving us no time to investigate any of the parameters we adopted in the current study. This is in agreement with the observations made in Punjab province of Pakistan, where vultures were healthy and in good condition with good body fat before they were found dead even when they were incubating the eggs (Riley et al., 2003) due to visceral gout. But no such observations of gout noticed in the present study. In this study, though we have used higher dose of the drug keeping in view the abnormal dose administered to the live stock in India by paraveterinarians and live stock owners, did not produce 100% mortality, which is, contradictory to the observations made by others (Lindsay Oaks and Martin, 2004) who used 2.5 mg/kg bwt which resulted in 100% mortality of vultures due to nephrotoxicity. We are of the opinion that those investigators have used only two vultures and that is not statistically significant number always. Our results indicated that, domestic fowl might be partially resistant to diclofenac compared to scavenging birds like vultures. Weight gain, haemoglobin, total leukocyte count, total erythrocyte count, packed cell volume (PCV) and total serum proteins have shown no significant changes in the values in any of the treatment, propylene glycol and control groups compared to the normal values reported for birds (Jain, 1993). Some researchers reported significant decrease in TLC values in Japanese quails when treated with furazolidone (Coles, 1986), but nimesulide, diclofenac and propylene glycol did not cause decrease in TLC in the present study.
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Creatinine and AST levels were significantly high in diclofenac group compared to the treatment and control groups indicating the probable muscular and hepatocellular damage caused by diclofenac. It is observed that the levels of creatinine and AST shoots up in case of hepatocellular and muscular damage (Lumeij, 1997; Lumeij and Waterhof, 1987), both values forms a good tool for assessing liver and muscle damage (Dabbert and Powell, 1993). In the present study, AST levels in the diclofenac group remained significantly elevated at the end of 28 days of experimentation. Our observations are in contradictory to the observations made by others (Kendel and Harr, 2002) where they observed elevated levels of AST for 100 h following doxycycline treatment and later dropped down to normal levels. Increased levels of AST were also noticed due to hepatocellular degenerative changes caused by furazolidone in Japanese quails (Nazifi and Asasi, 2001; Arbid et al., 1990). Serum cholesterol levels were found to be in normal range during the post-experimentation period of 28 days in nimesulide, vehicle control and control groups. Diclofenactreated group showed elevated levels of cholesterol probably indicating the renal damage (Coles, 1986). Elevated levels of cholesterol are usually observed in birds during vitellogenesis and egg formation (Johnson, 2000). But in the present study, abnormal serum cholesterol levels could be mainly due to renal damage but not due to vitellogenesis or egg formation, as all the birds used in the experimentation were of 6 weeks age. Alkaline phosphatase levels remained in normal levels as reported in some of avian species (Kendel and Harr, 2002; Simaraks et al., 2004) in all the treatment and control groups except diclofenac group. Elevated levels of ALP in diclofenac group are indicative of liver damage (Lumeij and Waterhof, 1987). The vehicle (propylene glycol,) control group did not show significant elevation of any of the parameters tested, which is in agreement with others who noticed that PG is non toxic to chicken (Hill and Camardese, 1981). All the above observations were supported by the altered architecture of kidney and liver sections upon histopathological examination after 28 days of post-treatment. However, we are not presenting all the histopathological data regarding spleen, heart, intestine and lungs, as there were no lesions observed in those organs. Kidney sections in diclofenac group showed, disruption of tubular architecture, lymphoid aggregation, intertubular fibrosis and marked congestion which is in agreement with, tubular necrosis and fibrosis observed in Oriental whitebacked vultures due to diclofenac poisoning (Lindsay Oaks and Martin, 2004). NSAIDs, including diclofenac, are also recognized as causing renal disease in mammals (Murray and Brater, 1993; Todd and Sorkin, 1998). Liver sections in diclofenac group showed, severe sinusoidal congestion with bileduct hyperplasia indicating liver injury caused by diclofenac sodium injection. To our knowledge, there was no published information available about adverse effects of nimesulide on haemopoetic system, liver, kidney muscle, intestine and spleen in avians. Hence, it was concluded that nimesulide and the vehicle (propylene gly-
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