CHEMOPROTECTIVE ROLES OF DIPHENYL DISELENIDE(DPDS) ON CHLORPYRIFOS(CPF)- INDUCED NEUROTOXICITY IN MID-BRAIN OF MALE RATS

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ABSRACT

Chlorpyrifos [CPF; O,O-diethyl O-(3,5,6-trichloro-2-pyridinyl) phosphorothionate] is a widely used organophosphate pesticide that is known to be toxic to the environment and to living beings. It affects several organs of the body especially the brain leading to impairment of normal functioning of the body. Diphenyl diselenide (DPDS), an organsoselenium compound is the simplest of the synthetic diaryldiselenides. Studies have shown that DPDS exhibits anti-inflammatory and antioxidant effects if administered in pharmacological doses. This present study was aimed at investigating the neurotoxic effects of CPF on the midbrain of male Wistar rats and the possible counter effects by DPDS.

This project work was carried out using a total of sixty (60) male Wistar rats which were grouped into five cages of twelve rats each. The animals underwent acclimatization before being treated for 5 weeks. Group A rats were given corn-oil as control; Group B rats received DPDS (5mg/kg) only dissolved in corn-oil; Group C rats were treated with CPF (5mg/kg) only dissolved in corn-oil; Group D rats were co-exposed to CPF (5mg/kg) + DPDS (2.5mg/kg) while Group E rats were co-exposed to CPF (5mg/kg) + DPDS (5mg/kg) for the whole of the 5 weeks after which they were euthanized. The mid-brain was excised, homogenized and centrifuged. Markers of brain oxidative damage and histopathology were evaluated in the rats.

Superoxide dismutase (SOD) activity, catalase (CAT) activity, reduced glutathione (GSH) level, Glutathione Peroxidase (GPx) activity, glutathione-S-transferase (GST) activity and acetylcholinesterase (AChE) activity were significantly(p < 0.05) reduced in the mid-brain of the rats treated with chlorpyrifos (CPF) alone as compared to the control rats. Also, oxidative and inflammatory markers such as hydrogen peroxide (H2O2) level, lipid peroxidation (LPO) and myeloperoxidase (MPO) activity respectively were significantly increased in the same rats. However, the co-exposure with DPDS ameliorated the neurotoxic effects of CPF by increasing the antioxidant level and suppressing the oxidative stress biomarkers. Also, the weights of the midbrain of rats treated with CPF showed no significant change.

Summarily, DPDS reversed the neurotoxic effects in midbrain induced by CPF via the antioxidant and anti-inflammatory properties which it possesses.

TABLE OF CONTENTS

Title pagei

Certificationii

Dedicationiii

Acknowledgementsiv

Table of contentsv

List of figuresix

List of tablesx

Abstractxi

CHAPTER ONE: INTRODUCTION AND LITERATURE REVIEW

1.1 Introduction1

1.1.1 Aim of study4

1.1.2 Objectives of the study4

1.2 Literature review5

1.2.1 The Nervous system5

1.2.2 The Brain 6

1.2.3 Structure and function of the brain7

1.2.3.1 The Brain stem7

1.2.3.2 Cerebrum 8

1.2.3.3 Cerebellum9

1.2.3.4 Hypothalamus9

1.2.3.5 Thalamus9

1.2.4 Chlorpyrifos (CPF)10

1.2.4.1 Neurological effects of CPF11

1.2.5 Physical and Chemical properties11

1.2.6 Toxicity of CPF12

1.2.7 Mechanism of action13

1.2.8 Selenium14

1.2.9 Diphenyl Diselenide (DPDS)14

1.2.9.1 Antioxidant action of DPDS15

1.2.9.2 Neuroprotective effects of DPDS16

1.2.9.3 Anti-inflammatory activity of DPDS16

1.2.10 Reactive oxygen species and free radicals17

1.2.10.1Superoxide anion19

1.2.10.2Hydrogen peroxide20

1.2.10.3Hydroxyl radical20

1.2.10.4Singlet oxygen21

1.2.11 Oxidative stress21

1.2.11.1Lipid peroxidation21

1.2.11.2Protein oxidation23

1.2.11.3DNA damage24

1.2.12 Antioxidant system24

1.2.12.1Antioxidant enzymes25

1.2.12.2Non-enzymatic antioxidants27

CHAPTER TWO:MATERIALS AND METHODS

2.1Chemicals29

2.2Experimental animals29

2.3Experimental design and treatment29

2.4Sacrifice of experimental animals30

2.5Homogenization30

2.6Reagents preparation31

2.7Biochemical assays31

2.7.1Determination of protein concentration31

2.7.2Determination of catalase34

2.7.3Assessment of lipid peroxidation35

2.7.4Estimation of reduced Glutathione GSH level37

2.7.5Determination of superoxide dismutase SOD activity41

2.7.6Determination of Hydrogen peroxide concentration42

2.7.7Estimation of Glutathione-S-Transferase activity46

2.7.8Assay for Glutathione peroxidase activity47

2.7.9Measurement of Myeloperoxidase activity49

2.7.10Determination of Acetylcholinesterase activity51

2.8Histopathology53

2.9Statistical analysis53

CHAPTER THREE: RESULTS

3.1Weight analysis54

3.2Biochemical analysis56

3.3Histopathological assessment65

CHAPTER FOUR: DISCUSSION AND CONCLUSION

4.1Discussion and Conclusion66

References 70

LIST OF FIGURES

Figure 1.1Anatomy of the Brain7

Figure 1.2 Structure of Chlorpyrifos10

Figure 1.3 Structure of Diphenyl diselenide14

Figure 2.1Standard curve for protein determination by Bradford’s method33

Figure 2.2MDA reaction in Lipid Peroxidation assay35

Figure 2.3Reaction of reduced GSH with Ellman’s Reagent 37

Figure 2.4GSH Standard curve40

Figure 2.5Calibration curve for Hydrogen Peroxide45

Figure 3.1Histopathogical photomicograph 65

LIST OF TABLES

Table 1.1Physical and chemical properties of Chlorpyrifos11

Table 1.2Clinical conditions involving reactive oxygen species18

Table 2.1GSH Standard Curve Protocol39

Table 2.2Preparation of H2O2 standard curve 43

Table 2.3Glutathione-S-Transferase Assay Medium47

Table 2.4 Protocol for Acetylcholinesterase activity52

Table 3.1Body weight change of male rats treated with CPF and DPDS 54

Table 3.2Brain weight change of male rats treated with CPF and DPDS55

Table 3.3GPx activity in mid-brain of male rats treated with CPF and DPDS 56

Table 3.4LPO level in mid-brain of male rats treated with CPF and DPDS57

Table 3.5CAT activity in mid-brain of male rats treated with CPF and DPDS58

Table 3.6SOD activity in mid-brain of male rats treated with CPF and DPDS59

Table 3.7GSH level in mid-brain of male rats treated with CPF and DPDS 60

Table 3.8GST activity in mid-brain of male rats treated with CPF and DPDS61

Table 3.9H2O2 level in mid-brain of male rats treated with CPF and DPDS62

Table 3.10MPO activity in mid-brain of male rats treated with CPF and DPDS63

Table 3.11AChE activity in mid-brain of male rats treated with CPF and DPDS64

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CHEMOPROTECTIVE ROLES OF DIPHENYL DISELENIDE(DPDS) ON CHLORPYRIFOS(CPF)- INDUCED NEUROTOXICITY IN MID-BRAIN OF MALE RATS

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