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Preliminary evaluation of an in vitro test for assessment of excitotoxicity by measurement of early gene (c-fos mRNA) levels

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Author: Griffiths, R. · Grieve, A. · Scollon, J. · Scott, M. · Williams, C. · Meredith, C.
Type:article
Date:2000
Institution: TNO Kwaliteit van Leven TNO Bibra
Source:Toxicology in Vitro, 5, 14, 447-458
Identifier: 280410
doi: doi:10.1016/S0887-2333(00)00034-5
Keywords: c-fos mRNA · Cerebellar granule cells · Early gene levels · Excitotoxicity · Predictive test · amino acid receptor blocking agent · biological marker · excitotoxin · glutamate receptor · glutamate receptor antagonist · messenger RNA · animal cell · controlled study · cytotoxicity · gene activation · gene expression · granule cell · mouse · nerve cell necrosis · nonhuman · oncogene c fos · toxicity testing · Animals · Cell Death · Cell Survival · Cells, Cultured · Cerebellar Cortex · Dose-Response Relationship, Drug · Evaluation Studies · Excitatory Amino Acid Antagonists · Gene Expression Regulation · Genes, fos · Glutamic Acid · Mice · Proto-Oncogene Proteins c-fos · RNA, Messenger · Animalia

Abstract

Using primary cultures of mouse cerebellar granule cells as an in vitro model system, it has been demonstrated that different profiles of temporal expression of the c-fos proto-oncogene are observed under non-excitotoxic and excitotoxic conditions. A ratio has been derived previously for the steady- state level of c-fos mRNA after 30 min and 240 min which suggests that a 240 min/30 min ratio of greater than 1 correlates with excitotoxicity, whereas a ratio of less than 1 correlates with a non-excitotoxic outcome. Moreover, a positive correlation is seen with abrogation of excitotoxicity in response to selective excitatory amino acid receptor antagonists. This test, proposed as a specific biomarker for excitotoxicity is undergoing prevalidation. Excitotoxicity is defined as neuronal cell death mediated by hyperactivation of glutamate receptor subtypes and therefore might be expected to be prevented by selective glutamate receptor antagonists. In preliminary evaluation studies, we have conducted work under the direction of the European Center for Validation of Alternate Methods (ECVAM) using compounds specified by ECVAM that have been subdivided into four groups based on known or presumed actions. These groups comprise: Group 1 - endogenous/synthetic excitotoxins; Group 2 - environmental, putative excitotoxins; Group 3 - neurotoxic but non-excitotoxic compounds, and Group 4 - non-toxic compounds. The results of this study support the proposal that the c-fos mRNA time-ratio test is a specific biomarker of excitotoxicity. Just as importantly, this test has the potential for application in screening newly-designed EAA receptor antagonists in the search for clinically relevant drugs to treat a variety of neuropathologies. (C) 2000 Elsevier Science Ltd. Chemicals/CAS: Excitatory Amino Acid Antagonists; Glutamic Acid, 56-86-0; Proto-Oncogene Proteins c-fos; RNA, Messenger