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

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 CONTENTSTitle pageiCertificationiiDedicationiiiAcknowledgementsivTable of contentsvList of figuresixList of tablesxAbstractxi
CHAPTER ONE: INTRODUCTION AND LITERATURE REVIEW1.1                  Introduction11.1.1            Aim of study41.1.2            Objectives of the study41.2                  Literature review51.2.1            The Nervous system51.2.2            The Brain 61.2.3            Structure and function of the brain71.2.3.1            The Brain stem71.2.3.2            Cerebrum 81.2.3.3            Cerebellum91.2.3.4 Hypothalamus91.2.3.5            Thalamus91.2.4            Chlorpyrifos (CPF)101.2.4.1            Neurological effects of CPF111.2.5            Physical and Chemical properties111.2.6            Toxicity of CPF121.2.7            Mechanism of action131.2.8            Selenium141.2.9            Diphenyl Diselenide (DPDS)141.2.9.1 Antioxidant action of DPDS151.2.9.2 Neuroprotective effects of DPDS161.2.9.3 Anti-inflammatory activity of DPDS161.2.10 Reactive oxygen species and free radicals171.2.10.1Superoxide anion191.2.10.2Hydrogen peroxide201.2.10.3Hydroxyl radical201.2.10.4Singlet oxygen211.2.11            Oxidative stress211.2.11.1Lipid peroxidation211.2.11.2Protein oxidation231.2.11.3DNA damage241.2.12            Antioxidant system241.2.12.1Antioxidant enzymes251.2.12.2Non-enzymatic antioxidants27 CHAPTER TWO:MATERIALS AND METHODS2.1Chemicals292.2Experimental animals292.3Experimental design and treatment292.4Sacrifice of experimental animals302.5Homogenization302.6Reagents preparation312.7Biochemical assays312.7.1Determination of protein concentration312.7.2Determination of catalase342.7.3Assessment of lipid peroxidation352.7.4Estimation of reduced Glutathione GSH level372.7.5Determination of superoxide dismutase SOD activity412.7.6Determination of Hydrogen peroxide concentration422.7.7Estimation of Glutathione-S-Transferase activity462.7.8Assay for Glutathione peroxidase activity472.7.9Measurement of Myeloperoxidase activity492.7.10Determination of Acetylcholinesterase activity512.8Histopathology532.9Statistical analysis53


CHAPTER THREE: RESULTS3.1Weight analysis543.2Biochemical analysis563.3Histopathological assessment65
CHAPTER FOUR: DISCUSSION AND CONCLUSION4.1Discussion and Conclusion66
References 70


















LIST OF FIGURESFigure 1.1Anatomy of the Brain7Figure 1.2 Structure of Chlorpyrifos10Figure 1.3 Structure of Diphenyl diselenide14Figure 2.1Standard curve for protein determination by Bradford’s method33Figure 2.2MDA reaction in Lipid Peroxidation assay35Figure 2.3Reaction of reduced GSH with Ellman’s Reagent    37Figure 2.4GSH Standard curve40Figure 2.5Calibration curve for Hydrogen Peroxide45Figure 3.1Histopathogical photomicograph 65

















LIST OF TABLESTable 1.1Physical and chemical properties of Chlorpyrifos11Table 1.2Clinical conditions involving reactive oxygen species18Table 2.1GSH Standard Curve Protocol39Table 2.2Preparation of H2O2 standard curve 43Table 2.3Glutathione-S-Transferase Assay Medium47Table 2.4 Protocol for Acetylcholinesterase activity52Table 3.1Body weight change of male rats treated with CPF and DPDS 54Table 3.2Brain weight change of male rats treated with CPF and DPDS55Table 3.3GPx activity in mid-brain of male rats treated with CPF and DPDS 56Table 3.4LPO level in mid-brain of male rats treated with CPF and DPDS57Table 3.5CAT activity in mid-brain of male rats treated with CPF and DPDS58Table 3.6SOD activity in mid-brain of male rats treated with CPF and DPDS59Table 3.7GSH level in mid-brain of male rats treated with CPF and DPDS 60Table 3.8GST activity in mid-brain of male rats treated with CPF and DPDS61Table 3.9H2O2 level in mid-brain of male rats treated with CPF and DPDS62Table 3.10MPO activity in mid-brain of male rats treated with CPF and DPDS63Table 3.11AChE activity in mid-brain of male rats treated with CPF and DPDS64