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Cognitive Tests Used for Evaluation of Neurotoxic Effects of Organic Solvent in Rats

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Cognitive Tests Used for Evaluation of Neurotoxic Effects of Organic Solvent in Rats Cognitive tests used for evaluation of neurotoxic effects of organic solvents in rats. Ph.D. Thesis Grete Ostergaard Roskilde Universitet Levnedsmiddelstyrelsen Department of Life Sciences National Food Agency of Denmark and Chemistry Institute of Toxicology DATA SHEET Title: Cognitive tests used for evaluation of neurotoxic effects of organic solvents in rats. Author: Grete 0stergaard Publisher: National Food Agency of Denmark Affiliation: Institute of Toxicology, Department of General Toxicology, National Food Agency of Denmark, Ministry of Health. Address: M@rkh@jBygade 19, DK-2860 Sgborg, Denmark Telephone: +45 39 69 66 00 Fax: +45 39 66 01 00 Please quote: Grete 0stergaard (1997): Cognitive tests used for evaluation of neurotoxic effects of organic solvents in rats. Ph.D. thesis. National Food Agency of Denmark, DK-2860, Soborg, Denmark. Keywords: Morris water maze, eight-arm radial maze, cognitive tests ISBN: 87-601-1745-l List of contents 1 PREFACE . 5 2 ACKNOWLEDGEMENTS . 6 3 LIST OF PAPERS INCLUDED . 7 4 OTHER PAPERS WITH RELATION TO THE SUBJECT OF THIS . 8 5 SUMMARY . 9 6 DANSK SAMMENDRAG . 10 7 INTRODUCTION . 11 8 ORGANIC SOLVENTS ........................... 14 8.1 Effects of solvents on the nervous system in man .................................. 15 8.2 Solvents involved in the development of chronic toxic encephalopathy ............... 16 8.3 Toluene ................................ 18 8.4 Effects of toluene in man ................... 18 8.5 Behavioural effects of toluene in laboratory animals ............................... 19 8.6 White spirit ............................ 23 8.7 Effects of white spirit in man ............... 23 8.8 Effects of white spirit in animals .............. 25 9 CHOICE OF EXPOSURE IN THE PRESENT PROJECT . 28 10 CHOICE OF TIME OF TESTING RELATIVE TO EXPOSURE IN THE PRESENT PROJECT . 29 11 BEHAVIOURAL TESTS IN THE PRESENT PROJECT ................................... 30 11.1 Theoretical types of memory ................ 30 11.2 Working memory and reference memory ...... 31 11.3 Effects of neurotoxic agents on learning and memory ............................... 34 11.4 Tests of learning and memory function ....... 34 11.5 Critical evaluation of cognition models in relation to the present project .............. 35 11.6 The eight-arm radial maze ................. 38 11.7 The Morris water maze ................... 47 12 REPORT OF OWN STUDIES ...................... 55 12.1 Eight-arm radial maze .................... 56 12.2 Morris water maze ....................... 61 12.3 Testing in radial maze and Morris maze ...... 64 13 RESULTS.. ................................... 67 13.1 Toluene ............................... 67 13.2 Aromatic white spirit ..................... 69 13.3 Dearomatized white spirit .................. 71 13.4 Summary of results ...................... 74 14 DISCUSSION .................................. 76 14.1 Demonstrated cognitive effects of organic solvents in our own laboratory ............. 80 14.2 Comparison of tests for cognitive function ...... 81 14.3 Endpoints and procedures in the eight-arm radial maze and the Morris water maze ....... 82 14.4 Testing in the present project ............... 83 15 CONCLUSION ................................. 86 16 REFERENCES ................................. 88 17 APPENDIX .................................... 97 Reference A ....................................... 128 Reference B ....................................... 136 Reference C ....................................... 142 1 PREFACE In 1985 a research project was initiated by the Danish Environmental Protection Agency with the aim to investigate the possible irreversible effects of long-term exposure to organic solvents in rats. The object was to obtain data that could be used for classification and labelling of organic solvents within the EU system. The ultimate goal was to protect workers and consumers from chronic toxic encephalopathy which is a toxin-induced symptom complex characterized by memory impairment, fatigue, mood disturbance, reduced intellectual capacity, and reduced ability to concentrate. This syndrome is thought to be caused by prolonged exposure to organic solvents, but it has not been possible to identify any individual chemicals as causative based on the available human data. Consequently, it has not been possible to label any particular organic solvent for this effect. The project attempted to identify solvents that after long-term inhalation exposure would produce persistent effects on the nervous system of experimental animals. In the project we included several different toxicological and neuro- toxicological disciplines to ensure a comprehensive examination of the exposed animals. The behavioural tests described in this Ph.D. thesis are thus only a small part of a multidisciplinary effort. 2 ACKNOWLEDGEMENTS I wish to thank Jfirgen Clausen, Institute of Life Sciences and Chemistry, University of Roskilde, Denmark, for accepting to be my supervisor. Ole Ladefoged, Department of General Toxicology at the Institute of Toxicology, is thanked for being my advisor. My project collaborators Peter Arlien-Soborg, Ulla Hass, Lise Korbo, Ole Ladefoged, Henrik Rye Lam, Jens-Jflrgen Larsen, S&-en Peter Lund, Arne Mgller, Kirsten Pilegaard, Leif Simonsen and Poul Strange are thanked for fruitful discussions and insight into the various disciplines of neurotoxicology. Lejf Burkal, Morten Falding, Ejgil Frank, Else-Marie Skov Jensen, Bo Larsen, and Bo Herbst provided excellent technical assistance throughout the project. Inger Bergmann and Lea Tobiassen from the Danish Environmental Protection Agency are thanked for their support and interest. Soborg, January 1997. Grete 0s tergaard 3 LIST OF PAPERS INCLUDED The present Ph. D. thesis covers the present monograph and is based upon the cognitive tests included in the papers A, B and C listed below. All the behavioural test systems (Morris water maze, eight-arm radial maze, passive avoidance test, motor activity test, functional observational battery test) have been elaborated and performed strictly by the present author and independently of the co-authors mentioned in the publications A, B, and C. References in the text are indicated by letters A-C. A. Ladefoged O., P. Strange, A. Moller, H.R. Lam, G. 0stergaard, J-J. Larsen, P. Arlien-Soborg (1991) Irreversible effects in rats of toluene (inhalation) exposure for six months. Pharmacology and Toxicology 68: 384-390. B. 0stergaard G., H.R. Lam, 0. Ladefoged, P. Arlien-Soborg (1993) Effects of six months’ white spirit inhalation exposure in adult and old rats. Pharmacology and Toxicology 72: 34-39. C. Lund S.P., L. Simonsen, U. Hass, 0. Ladefoged, H.R. Lam, G. Ostergaard (1996) Dearomatized white spirit inhalation exposure causes irreversible neurophysiological changes in rats. Neurotoxicology 18: 67-76. 7 4 OTHER PAPERS WITH RELATION TO THE SUBJECT OF THIS THESIS Bondy S.C., Lam H.R., 0stergaard G., Guo S.X., Ladefoged 0. (1995) Changes in markers of oxidative status in brain, liver and kidney of young and aged rats following exposure to aromatic white spirit. Arch Toxic01 69: 410-414. Korbo L., Ladefoged O., Lam H. R., 0stergaard G., West M. J., Arlien- Seborg P. (1996) Neuronal loss in hippocampus in rats exposed to toluene. NeuroToxicology 17: 359-366. Ladefoged O., Lam H.R., 0stergaard G., Hass U., Simonsen L., Lund S.P., Tobiassen L.S. (Eds.) (1996) Neurotoxicity testing of organic solvents in laboratory animals. TemaNord 53 1. Ladefoged O., Lam H-R., Ostergaard G., Nielsen E., Arlien-Soborg P.(1995) Neurotoxicology. Review of definitions, methodology and criteria. Miljopro- jekt nr. 282. Danish Environmental Protection Agency. Lam H.R., 0stergaard G., Guo S-X., Ladefoged O., Bondy S.C. (1994) Three weeks’ exposure of rats to dearomatized white spirit modifies indices of oxidative stress in brain, kidney, and liver. Biochem Pharmacol47: 65 l-657. Lam H.R., Ostergaard G., Ladefoged 0. (1995) Three weeks’ and six months’ exposure to aromatic white spirit affect synaptosomal neurochemistry in rats. Toxicology Letters 80: 39-48. Steensgaard A., 0stergaard G., Jensen C., Lam H.R., Topp S., Ladefoged O., Arlien-Sflborg P., Henriksen 0. (1996) Effect on the content of n- acetylaspartate, total creatine, choline containing compounds, and lactate in the hippocampus of rats exposed to aromatic white spirit for three weeks measured by NMR spectroscopy. Neurotoxicology (in press). 0stergaard G. (1995) Strain differences in radial arm maze performance of rats. Stand Journal of Lab Anim Sci 22: 229-235. 8 5 SUMMARY The aim of the present Ph.D. project was to develop a set of test methods for investigation of cognitive function in laboratory rats; and to use these methods in the evaluation of the neurotoxicity of selected organic solvents. A number of studies indicate that organic solvents may cause a syndrome known as chronic toxic encephalopathy in workers exposed to these chemicals. In the EU (European Union), protection of workers and consumers is effected through the use of regulatory classification and labelling of chemicals. In this system, it has not been possible to label solvents suspected of causing chronic toxic encephalopathy because the evidence present did not satisfy the criteria used in the classification guide. Therefore, a project was initiated by the Danish Environmental Protection Agency with the aim to investigate the possible irreversible effects of long- term exposure to organic solvents in rats. The study design, which had not been used before by other researchers,
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