Mercury, including its organic compounds, is a pervasive environmental and occupational neurotoxicant with multiple cellular targets of action. In addition to direct toxic effects on neurons, neurotoxicity results secondarily following impairment of other cell types that control the neuronal microenvironment, notably glia and the blood-brain barrier. Astroglia and cerebrovascular endothelial cells exhibit mutual trophisms in the expression of differentiated blood-brain barrier properties. These properties can now be reproduced in vitro in cell culture systems. Such in vitro systems offer an accessibility for quantitative analysis of cellular toxicity that is rarely afforded in situ. The ultimate objective of the proposed research is a detailed understanding of the effects of mercury on the selective permeability and transport properties of the blood-brain barrier and its cellular components, which would serve as a basis for new therapeutic interventions aimed at compensating or reversing such effects of mercury intoxication, or aimed at manipulating rates of mercury entry into or egress from the brain. A further long-term goal is the characterization and refinement of cell culture systems for their potential use in discriminating categories of toxicity to the blood-brain barrier. Specifically, the research will explore the selective impairment by mercuric chloride and methylmercury(II) chloride of characterized transport systems for anionic, neutral and cationic amino acids and glucose analogs in mouse cerebrovascular endothelial cells grown to confluence on porous membranes alone or 'enhanced' by co-culture with mouse cerebral astrocytes. Impairment of transport will be correlated with effects on the passive permeability of the endothelial cell layer, and with the cell content of mercury and its subcellular distribution. The role of serum constituents in modulating the migration of mercury into and across the endothelial cell layer will be explored. Radioactive tracers will be used to measure transport of amino acids and glucose analogs, and movement of mercury across the endothelial barrier. High-performance liquid chromatography will be used to measure endogenous free amino acid content in the cell cultures, and to validate the identify of radiolabeled species. Permeability of the cerebrovascular endothelial barrier will be assessed by measurement of electrical conductivity, and by spectrophotometric and fluorometric assays of molecular markers of varying bulk and charge. Scanning X-ray microanalysis of subcellular mercury distribution will be carried out using quick-freeze fixation and cryopreparative techniques.
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