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Mechanisms of m-dinitrobenzene-induced selective neurotoxicity

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  • Mechanisms of m-dinitrobenzene-induced selective

    neurotoxicity and the roles of brain glutathione

    A thesis submitted for the Degree of Doctor of Philosophy

    at the University of Leicester


    Henglong Hu

    MRC Toxicology Unit

    University of Leicester, UK

    December 1996

  • UMI Number: U106373

    All rights reserved

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  • Preface

    The translation of the word doctor from English to Chinese gives two distinct

    entities. One is used for medical professionals while the other for the Doctor of

    Philosophy. Having been called a doctor for many years, I became very enthusiastic

    in reading for a Doctor of Philosophy to make myself the other doctor. The desire to do

    so is not only for an extra title and another degree, but also for the purpose of being

    trained to a higher scientific standard, by following all the standard processes of PhD

    training in United Kingdom, a country which has made such a strong impact on modern

    science and technology.

    From experience I found that one attraction of scientific research is that a new

    phenomenon always turns up before the previous phenomenon has been fully

    understood. This may be what has been referred to as the challenge which keeps

    scientists interested and motivated. In the past three years, I enjoyed very much doing

    the proposed research programmes however at the same time, I also had the opportunity

    of enjoying exciting unexpected findings and looking into these interesting phenomena

    to a reasonable depth. I feel clearly that I am so small in the front of nature that it is

    impossible to look at everything in which I am interested, however small a specific

    research area is.

    Writing up this thesis is more or less telling the scientific stories that happened

    in this wonderful MRC laboratory during the past three years. This thesis is written and

    submitted for a Degree of Doctor of Philosophy, however I have no intention of

    implying that the project has been completed, simply because the more research is

    carried out, the more interesting questions turn up. Some pieces of work are quite pilot

    and preliminary although hopefully innovative.



    Preface 2

    Contents 3

    Abstract 8

    Abbreviations 9

    List of tables 11

    List of figures 12

    Acknowledgement 14

    Chapter 1 General introduction 15

    Neurotoxicology in general 15

    Functional or cellular selectivity of neurotoxicity 15

    Regional selectivity of neurotoxicity 18

    Oxidative stress in the central nervous system 19

    Metabolic capacity of brain towards xenobiotics 21

    Toxicities of m-dinitrobenzene (m-DNB) 24

    Working hypotheses and experimental strategy 26

    Chapter 2 Metabolism of m-dinitrobenzene (Literature review 1) 28

    Absorption and excretion of m-DNB after oral administration 28

    Distribution and pharmacokinetics of m-DNB in experimental

    animals 29

    Metabolic degradation of m-DNB in vivo and in vitro 30

    Further degradation of m-DNB metabolites 32

    Possible metabolic pathway of m-DNB 33

    Metabolism of m-DNB and free radical generation 34

    Chapter 3 Metabolism and functions of glutathione (GSH) in the central

    nervous system (CNS) {Literature review 2) 36

    GSH metabolism and its biological importance 36

    Regional distribution of GSH in brain 38

    Cellular, and subcellular distributions of GSH in the brain 39


  • Age dependent change of brain GSH levels 40

    Manipulation of brain GSH levels 41

    Brain GSH and ascorbic acid 42

    GSH and Nitric oxide 43

    GSH and mitochondrial functions 44

    Glutathione and neurodegenerative diseases 46

    Glutathione, NMDA receptor and CNS excitability 47

    Brain glutathione and behaviours 50

    Transport of glutathione 51

    Inborn errors of GSH synthesis 52

    Chapter 4 General materials and methods 53

    Animals and housing 53

    Rat models of DNB neurotoxicity 53

    Observations of clinical signs of neurotoxicity and

    behaviour changes 53

    General health checking 53

    Temperature control 54

    Ataxia scores 54

    Seizure observation and behaviour monitoring 54

    In vitro metabolic studies on tissue slices 54

    Brain and liver slice preparation and incubation 54

    Sampling and sample preparation for DNB measurement 55

    Detection and identification of m-DNB and its metabolites 55

    Implantation of intracerebroventricular (i.c.v.) cannula guides 56

    i.c.v. Administration of BSO and rat model of brain GSH depletion 56

    Preparation of regional brain samples for biochemical analysis 57

    High performance liquid chromatography (HPLC) measurement of

    brain GSH and relevant amino acids 57

    Derivatisation 57

    HPLC conditions 58

    Reagent preparation 58

    HPLC measurement of brain ascorbic acid and malondialdehyde 59


  • Pathological examination




    Chapter 5 Metabolism of m-dinitrobenzene by brain: in vitro studies 63

    Introduction 63

    Materials and methods 64

    Brain and liver slice preparation and incubation 64

    Sampling and HPLC sample preparation 64

    Detection and identification of m-DNB and its metabolites 65

    Statistics 65

    Results 66

    Tissue capacity for metabolising m-DNB 66

    Identification of metabolites and comparison of metabolic

    patterns 66

    Effects of DNB on glucose utilisation in brain slices 66

    Chemical reaction of nitrosonitrobenzene with GSH

    & ascorbate 66

    Discussion 68

    Chapter 6 Heterogeneity in regional distribution of glutathione, ascorbic

    acid and endogenous lipid peroxidation in rat brain 80

    Introduction 80

    Methods and Materials 82

    Results 84

    Heterogeneity of GSH and ascorbic acid distribution

    in the brain 84

    Heterogeneity of spontaneous lipid peroxidation in brain 84

    Heterogeneity of regional brain sensitivity to GSH depletion84

    Heterogeneity of GSH half lives across brain regions 84

    Effects of DNB on brain antioxidants and lipid peroxidation85

    Discussion 85

    Chapter 7 Age dependent changes of brain GSH and the regulation of GSH

    synthesis 95


  • Introduction 95

    Methods and Materials 95

    Results 96

    Developmental changes of Hippocampal GSH and cysteine 96

    Age-related changes of regional brain GSH levels 96

    Age-related changes of regional brain GSSG levels 96

    Age-related changes of regional brain cysteine level 96

    Discussion 96

    Age related profile of brain GSH 97

    GSH and development 97

    GSH and aging 97

    Regulation of brain GSH synthesis 98

    Usage of aged animal in toxicological studies 98

    Chapter 8 Brain glutathione status and m-dinitrobenzene neurotoxicity 104

    Introduction 104

    Methods and materials 105

    Implantation of i.c.v. cannula guide and BSO injection 105

    Rat models of DNB neurotoxicity 106

    Tissue preparation and HPLC measurement of brain GSH 106

    Ataxia scores 107

    Pathological examination 107

    Results 107

    Glutathione concentration in brain and its depletion 107

    DNB intoxication 108

    Discussion 109

    Chapter 9 Brain GSH modulation of central nervous system excitability 118

    Introduction 118

    Methods and materials 119

    Implantation of i.c.v. cannula guide

    and administration of BSO and y-GC 119

    Tissue preparation for biochemical analysis 120

    HPLC measurement of brain GSH and relevant amino acids 120


  • Pathological examination 120

    Seizure observation and behaviour monitoring 120

    Statistics 121

    Results 122

    Effects of BSO treatment on rat excitability 122

    Neurochemical changes after BSO treatment 122

    Discussion 124

    Chapter 11 General discussion 135

    Is oxidative stress responsible for m-DNB-induced neurotoxicity? 135

    Bioactivation of m-DNB in brain in situ and neurotoxicity 135

    Involvement of brain GSH in m-DNB-induced neurotoxicity 136

    Glutathione (GSH) in toxicology 138

    Regional and cellular selectivity of m-DNB neurotoxicity

    and brain antioxidants 140

    Coupling of oxidative stress with bioenergetic disorder in DNB

    neurotoxicity? 141

    Evidence for novel functions of GSH in CNS 142

    Conclusions 142

    Future work 143

    1. DNB metabolism and intracellular GSH distribution 143

    2. Further study on the oxidative stress and bioenergetic

    disorder in DNB-induced neurotoxicity 143

    3. CNS antioxidant system and brain susceptibility towards

    neurotoxins 144

    4. Metabolism and the importance of brain GSH 144

    5. Common factors and differences among the brain lesions

    produced by chlorosugars, acute thiamine deficiency, and

    neurotoxic nitro-compounds 145

    6. GSH involvement in

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