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Neurotoxicity of Toluene and Aromatic White Spirit

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Neurotoxicity of Toluene and Aromatic White Spirit NEUROTOXICITY OF TOLUENE AND AROMATIC WHITE SPIRIT A study of rat brain neurochemistry Ph.D. Thesis Henrik Rye Lam Institute of Toxicology Division of Biochemical and Molecular Toxicology Laboratory of Biochemical and Genetic Toxicology National Food Agency of Denmark and Institute of Life Sciences and Chemistry Roskilde University Center Denmark DATA SHEET Title: Neurotoxicity of toluene and aromatic white spirit. Subtitle: A study of rat brain neurochemistry. Author: Henrik Rye Lam. Publisher: National Food Agency of Denmark. Affiliation: Institute of Toxicology, Division of Biochemical and Molecular Toxicology, Laboratory of Biochemical and Genetic Toxicology. Address: Morkhoj Bygade 19, DK-2860 Soborg, Denmark. Telephone/fax: +45 39696600/+45 39660 100 Abstract: Male Wistar rats were inhalation exposed to 0, 500 (400), or 1500 (800) ppm toluene (aromatic white spirit) 6 h/day for 3 weeks (aromatic white spirit) or 6 months (toluene, aromatic white spirit). Regional and whole brain NA, DA, and 5-HT concentra- tions were statistically significantly changed by 6 months exposure to toluene. These changes were long-lasting. A 3 weeks aromatic white spirit study suggested that the threshold capacity for the elimination of aliphatic components was exceeded. Whole brain NA, DA, and 5- HT concentrations were statistically significantly increased. In a 6 months aromatic white spirit study, regional NA, DA, and 5-HT concentrations were statistically significantly changed. The level of 5-HT increased in 6 of 7 investigated regions. These changes were long-lasting. Following 3 weeks or 6 months of aromatic white spirit exposure, the yield of synaptosomal protein was statistically significantly reduced, whereas synaptosomal NA, DA, and 5-HT concentrations, high-affinity 5-HT uptake rate and uptake capacity were statistically significantly increased. Regional GFAP concentrations were not affected following 3 weeks or 6 months of aromatic white spirit exposure. The long-lasting toluene- and aromatic white spirit-induced changes of NA, DA, and 5-HT metabolism are serious findings which should be taken into consideration by regulators. Key words: Aromatic white spirit, brain regions, GFAP, neurochemistry, neurotoxicity, neurotransmitter metabolism, synaptosomes, toluene. Please quote: Henrik Rye Lam (1996): Neurotoxicity of toluene and aromatic white spirit. A study of rat brain neurochemistry. Ph.D. Thesis. National Food Agency of Denmark, DK- 2860, Soborg, Denmark. 138 pp. ISBN: 87-601-5591-4 Front cover: Section in the sagittal plane through the rat brain. PREFACE For several years, exposure to some organic solvents has been correlated with the induction of acute and long-lasting (i.e. long-term reversible or true irreversible) neurotoxicity in man. It is important to gain insight into this relationship in order to ensure sufficient protection of the population against these unwanted effects. Investigations in exposed laboratory animals can contribute to increase this knowledge and provide valuable information about the neurochemical basis of neurotoxicity in man. The present neurochemical studies constitute an integrated part of a collaborative multicenter neurotoxicology research project investigating the effects induced by organic solvents on the nervous system. The project was initiated in the mid-80s. Presently, it includes the following disciplines: general toxicology, behaviour, neuroelectrophysiology, neurochemistry, and neuropathology. The studies which constitute the experimental part of this thesis (Ladefoged et al., 1991; Lam et al., 1992; ostergaard et al., 1993; Lam et al., 1995) applied general toxicological, behavioural, clinical biochemical, neurochemical, neuropathological, and neurostereological approaches to study the effects induced by inhalation exposure of rats to toluene or aromatic white spirit. The neurochemical and clinical biochemical investigations were carried out by the author. Analysis of aromatic white spirit components in the rat brain was performed by Agneta Lof, Arbetslivsinstitutet. Solna, Sweden. Other collaborators contributed with the additional approaches: pathological and stereological studies were carried out by Ole Ladefoged, Arne Moller, and Poul Strange, and the behavioural investigations were carried out by Grete Q)stergaard and Jens-Jorgen Larsen. The neurochemical parameters include global, regional, and subcellular CNS neurotransmitter (noradrenaline, dopamine, and 5-hydroxytryptamine) metabolism and regional glial fibrillary acidic protein concentrations. Findings are discussed in terms of relevant neurochemical investigations published in the peer reviewed literature and in terms of the neurotoxicity of the 2 solvents in man. The applicability of the rat as a neurochemical research model for studying neurotoxicity in man is also discussed. I wish to thank professor Jorgen Clausen, Institute of Life Sciences and Chemistry, Roskilde University Center, Denmark, for accepting to be my supervisor and for his interest and fruitful comments during the writing phase. Thanks also to Jens-Jorgen Larsen, head of Department of General Toxicology at the Institute of Toxicology, for being my adviser. I am glad to make use of this opportunity to thank all project collaborators: Peter Arlien-Soborg, Stephen C. Bondy, Shirley X. Guo, Ulla Hass, Lise Korbo, Ole Ladefoged, Soren P. Lund, Agneta Lof, Arne Moller, K&ten Pilegaard, Leif Simonsen, Poul Strange, and Grete Qjstergaard for excellent collaboration within the various fields of neurotoxicology. Also thanks to Sven Edelfors and Dorrit Glendrup at the Department of Pharmacology, University of Copenhagen, for personal advice about the rat brain dissection procedure. I am grateful for the assistance of James P. O’Callaghan during the implementation phase of ‘his’ glial fibrillary acidic protein sandwich enzyme-linked immunosorbent assay technique and for the offered opportunity for inter-laboratory ‘calibration’ studies. I wish to thank Lejf Burkal, Morten Falding, Joan Gluver, Bo Herbst, Vibeke Kegel, Bo L. Jensen, Karen Roswall, Lonni Sand, and Berit Tonnesen for skilful technical assistance throughout the studies. Especially, I am in great debt to Annette Landin for more than 10 years of pleasant and productive, high-quality laboratory collaboration and for her sense of humour and patience, often needed to overcome episodes of pronounced stress. Other colleagues at the Institute of Toxicology and the National Food Agency of Denmark are thanked for help throughout the work. At last a thank to the Danish Environmental Protection Agency for support, interest, and encouragement in relation to the neurotoxicology research project. Soborg, May 1996. Henrik Rye Lam CONTENTS LIST OF PUBLICATIONS INCLUDED 1 ABBREVIATIONS 2 SUMMARY 4 DANSK SAMMENDRAG 7 I. INTRODUCTION 10 1. The nervous system 10 2. Neurotoxicity of chemical substances 12 3. Organic solvents 12 II. DEFINITION OF NEUROTOXICITY AND WORKING HYPOTHESIS 15 III. OBJECTIVES 16 TV. BASIC CHEMICAL, PHYSICAL, BIOCHEMICAL, AND NEUROTOXICOLOGICAL PROPERTIES OF TOLUENE AND WHITE SPIRIT 17 1. Toluene 17 2. White spirit 23 V. DRAWBACKS OF EPIDEMIOLOGICAL STUDIES. NEED AND LIMITATIONS FOR STUDIES IN LABORATORY ANIMALS 27 VI. STUDIES OF THE EFFECTS OF TOLUENE AND WHITE SPIRIT EXPOSURE ON BRAIN NA, DA, AND 5-HT METABOLISM 28 1. Toluene 28 2. White spirit 33 VII MATERIALS AND METHODS 34 1. Chemicals 34 2. Animals and exposure 34 3. Neurotransmitter metabolism 35 4. GFAP analysis 38 5. Protein analyses 39 6. Clinical biochemistry 39 7. Calculations and statistical analyses 39 8. Pathological and stereological methods 39 9. Behavioural methods 40 10. Analysis of aromatic white spirit components 40 VIII. RESULTS AND DISCUSSION 41 1. Toluene, reference I 41 2. Aromatic white spirit, reference II 56 3. Aromatic white spirit, reference III 56 4. Mechanisms potentially related to the generally increased 5-HT concentrations as observed in study II and III 57 5. Aromatic white spirit, reference IV 59 6. Overall conclusions on mechanisms underlying the demonstrated effects of aromatic white spirit exposure on rat brain neurotransmitter metabolism 62 7. Additional studies of the effect of aromatic white spirit exposure on regional rat brain GFAP concentrations 63 IX. INTERPRETATION OF THE NEUROCHEMICAL FINDINGS IN TERMS OF TOLUENE AND AROMATIC WHITE SPIRIT NEUROTOXICITY IN MAN 66 X. CONCLUSIONS 68 XI. REFERENCES 70 XII. APPENDICES 78 I A-D 79 II A-H 92 111A-B 104 XIII. OWN REFERENCES 107 Reference I 107 Reference II 115 Reference III 119 Reference IV 126 XIV. TABLES 137 Table I 137 Table II 138 LIST OF PUBLICATIONS INCLUDED This Ph.D. thesis is mainly based upon the neurochemical investigations as published in 4 research papers, which will be referred to throughout this report by their Roman bold numerals (I; II; III; IV): I Ladefoged, O., P. Strange, A. Mgller, H.R. Lam, G. astergaard, J.-J. Larsen & P. Arlien-Soborg: Irreversible effects in rats of toluene (inhalation) exposure for six months. Pharntacology & Toxicology 1991, 68, 384-390. II Lam, H.R., A. Liif & 0. Ladefoged: Brain concentrations of white spirit components and neurotransmitters following a three week inhalation exposure of rats. Pharmacology & Toxicology 1992, 70, 394-396. III (astergaard, G., H.R. L am, 0. Ladefoged & P. Arlien-Soborg: Effects of six months’ white spirit inhalation exposure in adult and old rats. Pharmacology & Toxicology 1993, 72, 34-39. IV Lam, H.R., G. astergaard & 0. Ladefoged: Three weeks’ and six months’ exposure to aromatic
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