1、 STD.API/PETRO PUBL 4b47-ENGL 1997 BB 0732290 U571130 47b BH American Institute Petroleum nE4“ sinyu /“ T documenting performance; and communicating with the public. API ENVIRONMENTAL MISSION AND GUIDING ENVIRONMENTAL PRINCIPLES The members of the American Petroleum Institute are dedicated to contin
2、uous efforts to improve the compatibility of our operations with the environment while economically developing energy resources and supplying high quality products and services to consumers. We recognize our responsibility to work with the public, the government, and others to develop and to use nat
3、ural resources in an environmentally sound manner while protecting the health and safety of our employees and the public. To meet these responsibilities, API members pledge to manage our businesses according to the following principles using sound science to prioritize risks and to implement cost-ef
4、fective management practices: 4 To recognize and to respond to community concerns about our raw materials, products and operations. 4 To operate our plants and facilities, and to handle our raw materials and products in a manner that protects the environment, and the safety and health of our employe
5、es and the public. 4 To make safety, health and environmental considerations a priority in our planning, and our development of new products and processes. e To advise promptly, appropriate officials, employees, customers and the public of information on significant industry-related safety, health a
6、nd environmental hazards, and to recommend protective measures. 4 To counsel customers, transporters and others in the safe use, transportation and disposal of our raw materials, products and waste materials. 4 To economically develop and produce natural resources and to conserve those resources by
7、using energy efficiently. 4 To extend knowledge by conducting or supporting research on the safety, health and environmental effects of our raw materials, products, processes and waste materials. To commit to reduce overall emission and waste generation. 4 To work with others to resolve problems cre
8、ated by handling and disposal of hazardous substances from our operations. 4 To participate with government and others in creating responsible laws, regulations and standards to safeguard the community, workplace and environment. 4 To promote these principles and practices by sharing experiences and
9、 offering assistance to others who produce, handle, use, transport or dispose of similar raw materials, petroleum products and wastes. STD.API/PETRO PUBL Lib47-ENGL 3997 0732290 0573332 249 E Brain Glial Fibrillary Acidic Protein (GFAP) as a Marker of Neurotoxicity During Inhalation Exposure to Tolu
10、ene Health and Environmental Sciences Department API PUBLICATION NUMBER 4647 PREPARED UNDER CONTRACT BY: HUGH L. EVANS, PH.D. NEW YORK UNIVERSITY MEDICAL CENTER NELSON INSTITUTE OF ENVIRONMENTAL MEDICINE TUXEDO, NEW YORK 10987 JUNE 1997 American Petroleum Ins titute STD-API/PETRO PUBL 4b47-ENGL L777
11、 B 0732270 057LL33 185 = FOREWORD API PUBLICATIONS NECESSARILY ADDRESS PROBLEMS OF A GENERAL NATURE. WITH RESPECT TO PARTICULAR CIRCUMSTANCES, LOCAL, STATE, AND FEDERAL LAWS AND REGULATIONS SHOULD BE REVIEWED. API IS NOT UNDERTAKING TO MEET THE DUTIES OF EMPLOYERS, MANUFAC- TURERS, OR SUPPLIERS TO W
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13、ISE, FOR THE MANU- FACTURE, SALE, OR USE OF ANY METHOD, APPARATUS, OR PRODUCT COV- ERED BY LETTERS PATENT. NEITHER SHOULD ANYTHING CONTAINED IN ITY FOR I“GEMENT OF LETTERS PATENT. THE PUBLICATION BE CONSTRUED AS INSURING ANYONE AGAINST LIABL- A11 rights reserved. No parr of this work may be reproduc
14、ed, stored in a retrieval system, or transmitted by any means, electronic, mechanical, photocopying, recording, or otherwise, without prior written permission from the publishe,: Contact the publisher, API Publishing Services, 1220 L Streer, N. W. Washington, D.C. 20005. Copyright Q 1997 American Pe
15、troleum Institute iii STD.API/PETRO PUBL qbi7-ENGL 1777 E 11732270 0573334 O33 9 ACKNOWLEDGMENTS THE FOLLOWING PEOPLE ARE RECOGNIZED FOR THEIR CONTRIBUTIONS OF TIME AND EXPERTISE DURING THIS STUDY AND IN THE PREPARATION OF THIS REPORT: API STAFF CONTACTS Dr. Robert Drew, Health and Environmental Sci
16、ences Department David Mongillo, Health and Environmental Sciences Department MEMBERS OF THE NEUROTOXICOLOGY TASK FORCE Wayne Daughtrey, Exxon Biomedical Sciences, Inc Charles Ross, Shell Oil Company Ceinwen Schreiner, Mobil Business Resources Corporation Christopher Skisak, Pennzoil Company MEMBERS
17、 OF THE NEW YORK UNIVERSITY MEDICAL CENTER WORK GROUP Technical assistance at New York University was provided by Zhaolong Gong, Dawn Gray, Alvin Little, Kenneth Magar, and Dr. Cheng Wang. Dr. Hassan El-Fawal contributed to the planning and interpretation of the GFAP assay. Dr. Bernard Jortner provi
18、ded neuropathology studies of our specimens in his laboratory at Virginia Polytechnical University. Dr. J. P. OCallaghan of the United States Environmental Protection Agency provided helpful suggestions on the GFAP method. Dr. Udai Singh assisted with the assays of corticosterone. Dr. Carroll Snyder
19、 contributed to the inhalation exposure methods. Dr. Ronald W. Wood and Dr. John G. Graefe provided the system for measurement of locomotor behavior during inhalation exposure. Supported in part by an Environmental Health Science Center Grant at NYU Medical Center (EH-00260). iv ABSTRACT Glial fibri
20、llary acidic protein (GFAP) was measured during and after sub-chronic exposure to toluene. Rats received inhalation exposure to air or 100 - 3,000 ppm toluene, 6 hr/day, 5 days/wk for up to 42 days. Toluene, in concentrations that are low for the rat (1 O0 to 1,000 ppm), altered GFAP and motor behav
21、ior without affecting body weight or producing overt signs of neurotoxicity. However, the declines in GFAP concentration during toluene exposure differ from the more commonly reported toxicant-induced pattern of increased GFAP. At a higher concentration (3,000 ppm), toluene produced increased GFAP c
22、oncentrations, observable neurological signs and weight loss. These results are discussed in relation to methodological issues and the relevant scientific literature. GFAP can provide an index of toxicity, even with exposures below the level which produce overt signs of toxicity. For toxicity screen
23、ing with animals, a battery including GFAP as well as behavioral and neurochemical measures would be useful. Implications for future research are discussed. STD.API/PETRO PUBL 4b47-ENGL 1777 SlS O732270 057113b 774 TABLE OF CONTENTS Section m EXECUTIVE SUMMARY .ES- 1 1 . INTRODUCTION 1 . 1 . 2 . MET
24、HODS 2-1 A“VfALS . 2-1 EXPOSURE TO TOLUENE . 2-1 BODY WEIGHT . 2-2 THYMUS AND ADRENAL GLAND WEIGHT . 2-2 LOCOMOTOR BEHAVIOR 2.2 NEUROPATHOLOGY . 2.3 . TOTAL PROTEIN IN THE BRAIN . 2-3 GFAP 2-4 CORTICOSTERONE 2-6 STATISTICS 2-7 . 3 . RESULTS 3-1 BODY WEIGHT . 3-1 THYMUS AND ADRENAL WEIGHT 3-1 BEHAVIO
25、R DURING TOLUENE INHALATION . 3-1 BEHAVIOR AFTER TOLUENE EXPOSURE . 3-2 NEUROPATHOLOGY . 3-3 QUALITY CONTROL: VARIABILITY IN PROTEIN DATA . 3-3 BRAIN TOTAL PROTEIN . 3-5 BRAIN GFAP 3-5 C ORT1 CO S TERONE 3 . 8 4 . DISCUSSION 4-1 IMPLICATIONS FOR FUTURE RESEARCH 4-5 REFERENCES R. 1 Figure 1. 2. 3. 4.
26、 5. 6. 7. 8. - STD.API/PETRO PUBL L(bLI?-ENGL 1997 9 0732290 057LL37 821 II LIST OF FIGURES The Replication of GFAP Assay Results . 2-6 Behavior during Toluene Inhalation and Post-exposure . .3-3 Significant Changes in Brain GFAP after the Third Day of Exposure to Toluene 3-6 Increased GFAP in the H
27、ippocampus of Rats Exposed for 3 and 7 Days Effects of 21 Days Exposure to 100,300 or 1,000 pprn Toluene to 3,000 ppm Toluene 3-6 on GFAP in the Hippocampus . 3-7 GFAP in the Cerebellum during 42 Days Exposure to 300 ppm Toluene . 3-8 to 1,000 ppm Toluene 3-8 and Serum Corticosterone after 3 and 7 D
28、ays Exposure to 1,000 ppm Toluene . 3-9 GFAP in the Cerebellum Returned to Baseline after 42 Days of Exposure Reduction in Thalamic GFAP on Days 3 and 7 of Exposure to 1,000 ppm Toluene LIST OF TABLES Table 1. 2. Body Weight during Exposure to Toluene 3-1 Summary of Effects on GFAP . 3-10 STD-APIIPE
29、TRO PUBL VbV-ENGL 1777 0732270 0573338 7b7 II EXECUTIVE SUMMARY Measures of brain cell-specific proteins show promise as markers of neurotoxicity in animals, particularly after exposure to heavy metals. One such marker is glial fibrillary acidic protein (GFAP). Increased GFAP indicates reactive glio
30、sis following neuronal injury from toxic exposures. Modern biochemical techniques for measurement of GFAP may prove to be faster, less expensive and more quantitative than classical neuropathological examination, and thus may be useful for evaluating potential neurotoxins. The purpose of this study
31、was to determine whether an immuno-assay for GFAP in the rats brain can provide practical evidence of toluene- induced neurotoxicity. The U.S. Environmental Protection Agency (USEPA, 1994, 1995) has suggested that a Radio-Immune-Assay (RIA) of brain GFAP be used in the screening for neurotoxicity of
32、 chemicals. Previous findings reported to API that an Enzyme-Linked-Immuno- Sorbant Assay (ELISA) of GFAP yielded results similar to results from the older RIA method and that the ELISA was sensitive to repeated oral exposure to lead (Pb) at exposure levels which produced behavioral and histological
33、 evidence of neurotoxicity (Evans, 1994a). The ELISA method has two advantages over the RIA method: freedom from radioactive materials, and simplicity. Although GFAP was a useful marker of Pb-induced rieurotox icity, GFAP was a less useful marker of Pb gosure than traditional indices such as blood l
34、ead concentration (Evans, 1994a). Toluene was chosen as a model neurotoxicant for these studies because its neurotoxicity in the rat has been characterized. The present studies documented changes in GFAP concentration during subacute inhalation exposure to toluene. Adult male F344 rats, at approxima
35、tely 47 days of age, received inhalation exposure to room air or 100,300, 1,000 or 3,000 ppm toluene, 6 hdday, 5 days/wk for up to 42 days. These exposures approximate an occupational exposure schedule. During and after exposure, the concentration of GFAP was determined in four brain regions. These
36、changes in GFAP were compared with standard neurotoxicity criteria: behavioral or neuropathological changes. Body weight was monitored as a sign of general toxicity. ES-1 - STD-APIIPETRO PUBL Lib47-ENGL 3777 m 0732270 U573337 bT3 The toluene concentration-effect data for GFAP concentration suggest t
37、hat 50% of brain samples are affected by an exposure of at least 3 days to 1,000 ppm toluene. At concentrations that are quite low with respect to the literature on the laboratory rat (100 to 1,000 ppm), toluene altered GFAP concentration without affecting body weight, brain pathology or producing o
38、vert signs of neurotoxicity. Changes in GFAP were seen as early as the third day of exposure; however, the declines in GF AP concentration differ from the more commonly reported toxicant-induced pattern of increased GFAP. GFAP was affected by toluene concentrations as low as 1 O0 ppm, within the ran
39、ge of occupational exposures for humans. In contrast, a much higher concentration (3,000 ppm) of toluene impeded growth and caused observable neurological signs in the rats, confirming previous reports of toluenes toxicity at high concentrations. Increased GFAP after 7 days exposure to 3,000 ppm is
40、suggestive of reactive gliosis, but cellular damage was not investigated at 3,000 ppm. At 1,000 ppm, cellular damage could not be seen at the light microscopic level. The time-effect data suggest that, as toluene exposure continued, significant changes in GFAP appeared, then reversed as exposure dur
41、ation continued. There was no evidence of permanent nervous system damage or functional impairment. For example, significant increases in GFAP at 42 days of exposure to 1,000 ppm toluene had returned to control levels by 14 days after exposure. No behavioral changes could be detected in the home cag
42、e in the 24 hours after the most recent exposure. The information provided by GFAP is partly correlated with, but not redundant to, that available from standard assays of behavior and general signs of toxicity such as body weight. GFAP was clearly more sensitive to toluene than histopathology indica
43、tes at the light microscopic level. GFAP was nearly equal to the sensitivity of behavioral measures, keeping in mind that the most sensitive behavioral index was recorded during toluene inhalation, whereas GFAP was measured 24 hours or more after the last exposure, at a time when behavior in the hom
44、e cage and neuropathological indices were unaffected. GFAP was of similar sensitivity to physiological ES-2 indices of inhaled toluene, as reported in the literature. The most sensitive indices at present are those reflecting changes in brain neurotransmitter function. Because the direction of chang
45、es in GFAP concentration was inconsistent as repeated toluene exposure continued, GFAP alone may not provide a practical marker of the effects of short term occupational exposure to toluene. Measurement of GFAP concentration with an ELISA should be an element in toxicity screening batteries, along w
46、ith behavior and indices of neurotransmitter function. An inter-laboratory workshop would be useful to advance the standardization of the GFAP ELISA. Further research is needed to identi how toxicant-induced changes in GFAP concentration are influenced by sub-types of astrocytes, changes in expressi
47、on of the GFAP gene, adrenal cortical steroid production, or neurotransmitter function. ES-3 Section 1 INTRODUCTION The nervous system is a target organ for inhaled toluene (ATSDR, 1994; Morata et al., 1995) and for many other organic solvents (Arlien-Soborg, 1992). Exposure to solvents has been all
48、eged in neurobehavioral disorders. The cellular and molecular mechanisms by which inhaled toluene causes changes in function of the central nervous system are not well understood (ATSDR, 1994). This is not surprising, since little more is known about the cellular and molecular mechanisms of action o
49、f inhaled volatile anesthetics, despite the routine use of those chemicals in human surgery (Pocok and Richards, 1993; Snyder and Andrews, 1996). The detection of the effects of inhaled toluene, and an understanding of the mechanisms of neurotoxicity, may be indicated by molecular markers. Solvents and their metabolites are cleared rapidly from the body (Brugnone et al., 1995) and markers of solvent neurotoxicity have not been validated for peripheral media, e.g., blood, urine (ATSDR, 1994; Tardif et al. , 1991). Most promising as markers of neurotox
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