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
By
Henglong Hu
MRC Toxicology Unit
University of Leicester, UK
December 1996
UMI Number: U106373
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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.
2
CONTENTS
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
3
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
4
Pathological examination
Statistics
59
60
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
5
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
6
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
