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EFFECTS OF VARIED ORAL DOSES OF TRIMETHOPRIM-SULFAMETHOXAZOLE THERAPY ON THE THYROID GLANDS, HAEMATOLOGY, SERUM CHEMISTRY AND SPERM COUNT IN NIGERIAN LOCAL DOGS
ABSTRACT
The effects of varied oral doses of trimethoprim-sulfamethoxazole therapy on the thyroid glands, haematology, serum chemistry and sperm count were evaluated in Nigerian local dogs. Twenty adult male dogs weighing between 4.5-10 kg were used for the study. The dogs were housed in metal cages in the Animal house of Department of Veterinary Parasitology and Entomology, University of Nigeria, Nsukka. The dogs were acclimatized for three weeks during which period they were vaccinated against rabies and screened for haemoparasites, helminthes and ectoparasites. The dogs were randomly assigned to four groups (A-D) of five dogs each. Baseline parameters were taken before the commencement of the experiment. Dogs in group B received 30mg/kg body weight (b.w) of trimethoprim- sulfamethoxazole (T/SMX) in drinking water. Dogs in group C received 60mg/kg b.w of T/SMX in drinking water whereas dogs in group D received 120mg/kg b.w of T/SMX in drinking water. Dogs in group A served as the control and received only clean drinking water. Treatment with T/SMX was at 12-hour intervals for a period of 21 days. About 5ml of blood was withdrawn weekly through the cephalic vein of all the dogs for haematology, serum enzyme assay and serum thyroid hormone assay. The rectal temperature, body weight, packed cell volume (PCV), haemoglobin concentration (HbC), red blood cell (RBC) count, total white blood cell (TWBC) count, serum enzymes-aspartate aminotransferase (AST), alanine amonotransferase (ALT) and alkaline phosphatise (ALP) activities, serum triiodothyronine (T3), thyroxine (T4), and serum thyrotropin (TSH) levels of all the dogs were assayed weekly. At the end of the 21-day treatment period, the dogs were humanely sacrificed. The thyroid glands, liver, testes and epididymis were dissected. Epididymal sperm count (ESC) was determined for each dog following standard procedures. Both the thyroid and liver were observed for gross pathological changes. Tissue sections were taken from both organs and processed histologically following standard procedures. They were observed for pathological changes under the light microscope. Data generated from this study were subjected to one-way analysis of variance. Variant means were separated using the least significant difference method. Significance was accepted at P < 0.05.
The thyroid glands of groups C and D dogs showed petechial and ecchymotic haemorrhage grossly. At microscopy, follicular cell hypertrophy, colloid depletion and haemorrhage were observed in groups C and D dogs, while group B dogs showed only colloid depletion. The liver of groups B, C and D dogs showed multifocal areas of necrosis and widespread vacuolar degeneration. The PCV (%) of group D dogs (32.06 ± 3.90) was significantly (p < 0.05) lower than that of group A dogs (44.40 ± 4.14) on days 14 and 21 of treatment. The HbC (g/dl) of group D dogs (11.72 ± 0.13) was significantly (p < 0.05) lower than that of group A dogs (14.76 ± 0.32) on days 14 and 21 of treatment. The RBC counts (x106 cells/mm3) of groups B (6.00 ± 0.26), C (5.24 ± 0.37) and D dogs (4.37 ± 0.17) were significantly (p < 0.05) lower than that of group A dogs (7.38 ± 0.74) on days 14 and 21 of treatment. These suggested normocytic normochromic anaemia. The TWBC counts (x103 cells/mm3) of groups B (6.16 ± 0.81), C (8.22 ± 0.87) and D dogs (6.90 ± 1.20) were significantly (p < 0.05) lower than that of group A dogs (11.34 ± 1.08) on day 7. There were no significant (p > 0.05) variations in the serum AST activities across the groups. However, serum ALT activity (IU/L) of the group D dogs (16.73 ± 3.77) was significantly (p < 0.05) higher than that of group A dogs (8.98 ± 1.87) on days 14 to 21, while serum ALP activity (IU/L) of group D dogs (161.00 ± 19.42) was significantly (p < 0.05) higher than that of group A dogs (72.89 ± 5.96) on days 14 and 21 of treatment. Serum T3 levels (ng/dl) of group C dogs (0.32 ± 0.05) was significantly (p < 0.05) lower than that of group A dogs (0.63 ± 0.07) on day 21 of treatment. Serum T4 levels of groups B (0.16 ± 0.09), C (0.21 ± 0.01) and D (0.03 ± 0.03) dogs were significantly (p < 0.05) lower than that of group A dogs (0.36 ± 0.05) on days 14 and 21. Serum TSH (µIU/L) of group D dogs (0.64 ± 0.13) was significantly (p < 0.05) higher than that of group A dogs (0.09 ± 0.06) on day 21 of treatment. Serum testosterone (ng/ml) level of group D dogs (4.97 ± 0.80) was significantly (p < 0.05) higher than that of group A dogs (0.76 ± 0.64) on day 14 of treatment. The ESC of groups B (0.88 ± 0.18) and C (0.47 ± 0.07) dogs were significantly higher than that of group A (0.40 ± 0.01), while that of group D dogs (0.06 ± 0.01) was significantly (p < 0.05) lower than that of group A.
In conclusion, this study demonstrated that T/SMX therapy as used in this study severely affected thyroid morphology and function, suppressed erythropoiesis , caused mild leukopenia, caused hepatocyte necrosis and elevation of serum ALT and ALP activities and serum testosterone level and affected epididymal sperm count in Nigerian local dogs. Therefore prolonged use (for 21 days) of this drug at high doses should be discouraged. Male dogs treated with T/SMX for up to 21days may not be fit for breeding due to possible scrotal lesions and low sperm count. Treatment with T/SMX should be accompanied with folic acid and vitamin E to reduce the oxidative damage to tissues associated with its use.
TABLE OF CONTENTS
Title page i
Certification and Approval ii
Dedication iii
Acknowledgements iv
Table of contents v
List of Tables vi
List of Figures vii
Abstract viii
CHAPTER ONE: INTRODUCTION
1.1 Introduction 1
1.2 Statement of the Problem 3
1.3 Aims and Objectives 4
CHAPTER TWO: REVIEW OF RELATED LITERATURE
2.1 Sulfonamides and Sulfonamide Combinations 5
2.1.1 Spectrum of Activity of Sulfonamides 5
2.1.2 Mechanism of Action of Sulfonamides 6
2.1.3 Categories of Sulfonamides 6
2.1.4 Potentiated Sulfonamides 7
2.1.5 Toxicity and Adverse Reactions to Sulfonamides 8
2.1.5.1 Keratoconjunctivitis Sicca 9
2.1.5.2 Nonregenerative Anaemia 9
2.1.5.3 Iatrogenic Hypothyroidism 10
2.1.5.4 Crystallization in Urine 10
2.1.5.5 Idiosyncratic Toxicosis 11
2.2 The Thyroid Gland 11
2.2.1 Organogenesis and Histology 11
2.2.2 Thyroid Hormone Biosynthesis 13
2.2.3 Thyroid Hormone Secretion 13
2.2.4 The Negative feed-back Mechanism 14
2.2.5 Functions of Thyroid Hormones 15
2.2.6 Thyroid Hormone Metabolism 16
2.2.7 Mechanisms of Thyroid Insufficiency 17
2.2.7.1 Blockage of iodine uptake 17
2.2.7.2 Thyroid peroxidase Inhibition 17
2.2.7.3 Blockage of Thyroid Hormone Release by Excess Iodide and Lithium 18
2.2.8 Response of the Thyroid Gland to Injury 19
2.2.8.1 Follicular Cystic Hyperplasia 19
2.2.8.2 Focal or Multifocal Follicular Hyperplasia 19
2.2.8.3 Follicular Cell Adenoma 20
2.2.8.4 Follicular Cell carcinoma 20
2.2.9 Disorders of Thyroid Function 21
2.2.9.1 Hypothyroidism 21
2.2.9.2 Hyperthyroidism 22
2.2.10 Goitre: Goitrogenesis and Forms of Goitre 22
2.2.10.1 Diffuse Hyperplastic Goitre 23
2.2.10.2 Colloid Goitre 23
2.2.10.3 Nodular Goitre 24
2.2.10.4 Congenital (Dyshormonogenetic) Goitre 25
2.2.11 Methods of Evaluating Thyroid Function 25
2.2.11.1 Assay of Thyroid Hormones and Thyroid Stimulating Hormone 25
2.2.11.2 Haematology 26
2.2.11.3 Morphologic and Morphometric Evaluation of Thyroid Gland 26
CHAPTER THREE: MATERIALS AND METHODS
3.1 Materials 28
3.1.1 Animals 28
3.1.2 Trimethoprim-Sulfamethoxazole 28
3.1.3 Laboratory Equipment and Reagents 28
3.2 Methods 29
3.2.1 Experimental Design 29
3.2.2 Collection of Blood Samples 30
3.2.3 Parameters Assessed 30
3.2.3.1 Rectal Temperature 30
3.2.3.2 Morphometric Studies 31
- Body Weght 31
- Thyroid Weight 31
- Testicular Weight 31
- Organosomatic Index 31
3.2.3.3 Haematology 32
- Packed Cell Volume 32
- Haemoglobin Concentration 32
- Red Blood Cell Count 33
- Total White Blood Cell Count 33
- Differential White Blood Cell Count 33
3.2.3.4 Serum Enzyme Assay 34
- Serum Aspartate-aminotransferase 34
- Serum Alanine-aminotransferase 35
- Serum Alkaline Phosphatase 35
3.2.3.5 Hormonal Assay 36
- Procedure for Serum T3 and T4 Assay 36
- Procedure for Serum TSH Assay 37
- Procedure for Serum Testosterone Assay 37
3.2.3.6 Epididymal Sperm Reserves 38
3.2.3.7 Gross and Histopathology 38
3.2.3.8 Data Analysis 39
CHAPTER FOUR: RESULTS
4.1 Clinical Signs and Physical Observations 40
4.2 Rectal Temperature 40
4.3 Morphometric Studies 40
4.4 Haematology Results 41
4.5 Serum Enzyme Assay Results 43
4.6 Hormonal Assay 43
4.7 Epididymal Sperm Count 44
4.8 Gross Pathology of the Thyroid and Liver 44
4.9 Histopathology 45
CHAPTER FIVE: DISCUSSION, CONCLUSION AND RECOMMENDATIONS
5.1 Discussion 82
5.2 Conclusion and Recommendations 88
REFERENCES 90
APPENDICES 104
CHAPTER ONE
1.1 INTRODUCTION
Diseases of the endocrine system are common especially in small animals. The thyroid is the largest of the endocrine organs that function exclusively as endocrine glands and is responsible for the synthesis, storage and secretion of thyroid hormones, triiodootyronine (T3) and thyroxine (T4) (Krista, 2012). Thyroid hormones regulate normal body growth in the young, maintain skeletal maturation, and regulate body temperature and various metabolic processes in the body including that of carbohydrate, protein and fat, and generally maintaining the basal metabolic rate (BMR) (Goglia et al., 2002).
The two most common problems with the thyroid gland are hypothyroidism and hyperthyroidism. However, some therapeutic agents have been associated with thyroid dysfunction either in vivo or in vitro and these include glucocorticoids, diphenylhydantoin, propylthiouracil, phenobarbital, phenylbutazone, furosemide, sulfonylureas and iodine-containing radio-contrast agents (Feldman and Nelson, 1987; Ferguson, 1989). Many reports have also linked sulfonamides and their potentiated forms such as trimethoprim-sulfomethoxazole to clinical hypothyroidism in animals (Hall et al., 1993; Torres et al., 1996; Brenner et al., 2009). However, their anti-thyroid activities vary among species (Panciera and Post, 1992).
Sulfonamides are broad spectrum antimicrobial agents that inhibit gram positive and gram-negative bacteria as well as protozoa such as coccidia (Appelgate 1983; Prescott and Baggot, 1993). Since the discovery of sulfonamides they have played a major role in the chemo-therapeutic control of infections, and their usefulness in veterinary medicine cannot be overemphasized (Mandell and Sande, 1990). The therapeutic efficacy plus the ease of administration and relatively low cost make these drugs very valuable agents in medical practice. However, the serious disadvantage of sulfonamides is the toxic reactions which occasionally occur during their use. Some of the toxic reactions to sulfonamides include crystals formation in the kidney especially in young dehydrated animals, keratoconjuctivitis sicca in dogs evidenced by increased sensitivity to light, corneal ulceration, and hepatotoxicity in dogs (18-53mg/kg trimethoprim-sulfonamide), increased bleeding time, elevated blood urea nitrogen and creatinine, haemolytic anaemia, urticaria, hypothyroidism and aplastic anaemia (Roder, 2004). These toxic reactions may occur even on administration of therapeutic doses in some species (idiosyncrasies), but are seen more frequently when high doses are given and/or administration is prolonged. The need for prolonged administration may occur in certain disease process such as staphylococcal pyoderma, in which medication is supposed to be continued for a minimum of 4 to 6 weeks after clinical resolution of pyoderma (Muller et al., 1989). On the other hand, the use of higher doses and the need for potentiation may have arisen due to widespread resistance by animal pathogens to sulfonamides (and trimethoprim also) as a result of many decades of therapeutic use (Riviere et al., 1991; Prescott and Baggott, 1993). In one study, prolonged treatment of dogs with potentiated sulfonamides at doses within the therapeutic range (15-30mg/kg body weight) produced no alteration in serum T3 andT4 (Panciera and Post, 1992). In another study, trimethoprim alone did not depress thyroid function, suggesting that the sulfonamide component of the combination drug may be the one responsible for lowering the thyroid hormone levels (Torres et al., 1996).
Idiosyncratic sulfonamide toxicity has been reported more in Doberman pinscher than in other breeds (Cribb, 1989; Cribb and Spielberg, 1990). On the other hand, iatrogenic hypothyroidism due to potentiated sulfonamides has been reported in Labrador and Golden Retrievers (Torres et al., 1996; Brenner et al., 2009), as well as in mixed breed of dogs (Gooking et al., 1999).
Many studies on sulfonamides toxicity make reference at times to thyroid pathology and dysfunction following an increased dose of the drug. Although hypothyroidism has been reported with sulfonamide use in dogs, it appears T3 is not affected unlike T4 (Hall et al., 1993). It is not clear whether the therapeutic dose range produces any toxic effect on the thyroid gland. Dogs treated with 16.7mg/kg of sulfamoxole and 3.3mg/kg of trimethoprim daily for six months were observed to have increased radioactive iodine uptake by the thyroid gland and a histological appearance compatible with increased thyroid activity and pituitary thyrotropin secretion (Lagler et al., 1976). A similar result was reproduced by the same author when the doses were increased to 50mg/kg and 10mg/kg respectively for four weeks.
1.2 STATEMENT OF THE PROBLEM
There are no reports in available literature of any untoward effects of trimethoprim-sulfamethoxazole combination drug therapy in Nigerian local dogs. Available data on other dog breeds are based mainly on assay of thyroid hormone levels in treated dogs with insufficient information on the possible histo-morphological changes in the thyroid tissues as already established in the rat model. Previous studies have also not evaluated the histopathological changes in the liver attributable to potentiated sulfonamide toxicity or potentiated sulfonamide-induced thyroid dysfunction. The effects of varied high doses of potentiated sulfonamide on the blood cells and integrity and function of some other vital organs like the liver and testes were considered worth investigating. It is therefore imperative to clarify through further research the effects of sulfonamide-trimethoprim drug combination on the thyroid glands of dogs.
1.3 AIMS AND OBJECTIVES
The overall aim of this study was to investigate the effects of varied oral doses of trimethoprim-sulfamethoxazole therapy on the thyroid gland, haematology, serum chemistry and sperm count in Nigerian local dogs. The specific objectives were to evaluate the effects of varied oral doses of trimethoprim-sulfamethoxazole therapy on the following parameters in Nigerian local dogs:
- Thyroid hormones (T3 and T4) and thyroid stimulating hormone (TSH) levels.
- Gross and histomorphology of the thyroid gland and liver.
- Haematology
- Serum biochemical profile
- Epididymal sperm count
EFFECTS OF VARIED ORAL DOSES OF TRIMETHOPRIM-SULFAMETHOXAZOLE THERAPY ON THE THYROID GLANDS, HAEMATOLOGY, SERUM CHEMISTRY AND SPERM COUNT IN NIGERIAN LOCAL DOGS
