Disruption of dopamine circuitry following exposure to the organochlorine insecticide endosulfan: Implications for neurological disease Public

Abstract

Abstract

Disruption of dopamine circuitry following exposure to the organochlorine insecticide endosulfan: Implications for neurological disease

Lauren Shapiro

Background: Endosulfan is an organochlorine insecticide and acaricide used to protect a wide variety of foods, plants, trees and shrubs. Although endosulfan was banned in 2011, its ability to persist in the environment and human body poses a threat to public health. Epidemiological studies have suggested that endosulfan exposure contributes to neurological disease, however there is minimal research that addresses the cellular mechanism or target of endosulfan in the nervous system.

Objective: This current study is designed to determine whether endosulfan exposure disturbs the dopaminergic system and contributes to the onset of neurological disorders.

Methods: Endosulfan toxicity was first assessed in the SH-SY5Y dopaminergic cell line using a cytotoxicity assay, and then in primary culture neurons taken from the ventral mesencephalon. Finally, the impact of endosulfan was evaluated using mice treated with 1 mg/kg of endosulfan for 30 days, mimicking potential human exposure. Animals were challenged with MPTP to evaluate endosulfan's impact on the dopaminergic system in the striatum. Immunohistochemistry and immunoblotting were performed to determine the effect of endosulfan on various neuronal proteins.

Results: Endosulfan caused toxicity in SH-SY5Y cells in a dose dependent fashion (LD50=400 uM). Primary culture neurons were drastically more sensitive to endosulfan as the LD50 in this model was 20 uM. The animal exposure study confirmed previous findings that MPTP decreases striatal dopamine transporter (DAT) and tyrosine hydroxylase (TH) levels by 66% and 31% respectively. Assessment of cortical dopaminergic markers revealed novel results that indicate endosulfan elicits a 35% decrease in DAT and a 50% decrease in TH. Finally, endosulfan exposure caused a 40% increase in cortical synapsin expression.

Discussion: Endosulfan's impact on DAT and TH indicates that exposure disrupts the dopaminergic system in the cortex. Synapsin is a protein essential for transmission in allneural systems, suggesting that an increase in cortical synapsin implicates changes to normal neural transmission in the cortex. Future studies must determine behavioral and neurochemical changes induced by endosulfan to fully elucidate its effect on the brain, and its association to neurological disease such as autism, ADHD and schizophrenia.

Table of Contents

TABLE OF CONTENTS

INTRODUCTION 1
Parkinson's Disease 1
Anatomy 2


Pathways 2

Treatment 3

Dopamine 3
Synthesis 3
Synaptic transmission 4
Receptors 4
Neural mechanism 5
Proteasomal dysfunction 5
Mitochondrial dysfunction 6
Oxidative stress 6
Gene-environment interaction 7
Genetic components 7
Environmental components 8
Identified risk factors 9
Metals 9
Pesticides 9
Fungicides 10
Herbicides 10
Insecticides 10
Organochlorines 10
Current study 12
Public health relevance 14
METHOD 15
Cytotoxicity assay 15
Primary culture 15
Culture preparation 15
Stereology 16
Animal study 16
Dosing 16
1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) 17
Western blots 17
Immunohistochemistry 18
Statistical analysis 18
RESULTS 18
Analysis of endosulfan toxicity in SH-SY5Y cell line 19
Analysis of endosulfan toxicity in primary culture 19
Analysis of endosulfan toxicity in adult mice 19

Striatal expression 20
Immunohistochemical analysis 20
Cortical expression 20
DISCUSSION 21
FIGURES 26
REFERENCES 37

FIGURES:
Figure 1.

Dopaminergic neuron: Synthesis, Packaging and Secretion 26
Figure 2. Cytotoxicity assay: Endosulfan toxicity in SH-SY5Y cells 27
Figure 3. Primary culture: Endosulfan toxicity in ventral mesencephalic neurons 28
Figure 4. Western blot analysis of striatal dopaminergicmarkers 29
Figure 5. Immunohistochemistry of striatal dopaminergic markers 31
Figure 6. Western blot analysis of striatal GABAergic markers 32
Figure 7. Western blot analysis of striatal synaptic markers and transporters 33
Figure 8. Western blot analysis of cortical dopaminergic markers 34
Figure 9. Western blot analysis of cortical GABAergic markers 35
Figure 10. Western blot analysis of cortical synaptic markers and transporters 36

About this Master's Thesis

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School	Rollins School of Public Health
Department	Environmental Health
Degree	MPH
Submission	Master's Thesis
Language	English
Research Field	Health Sciences, Public Health
Mot-clé	organochlorine, endosulfan, dopamine, brain, toxicology, neurotoxicology
Committee Chair / Thesis Advisor	Caudle, William Michael, Emory University
Committee Members	Tolbert, Paige, Emory University
Partnering Agencies	Emory University schools, faculty or affiliated programs

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