Two goals of Pb neurotoxicity research are to identify molecular-and cellular alterations that underlie behavioral deficits associated with low level exposure and to define mechanisms of Pb uptake and tolerance in cells that accumulate Pb. Cell cultures are practical tools with which to pursue these goals, offering an extracellular environment that can be precisely manipulated and direct observation. Both neurons and neuroglia are probable sites of Pb-induced damage in brain. Astroglia, a type of neuroglia, are proposed to serve as a Pb depot or filter in brain. In culture these cells accumulate Pb from the surrounding medium and store it intracellularly. As a non-physiological metal, Pb must gain entry into cells such as astroglia by subverting mechanisms that exist for the transport of other molecules. Two transport mechanisms have been identified: an anion exchanger in red cell ghosts and calcium channels in adrenal chromaffin cells. Neither of the above mechanisms for Pb entry has been studied in cultured astroglia, although both anion exchangers and Ca2+ channels are found in these cells. In view of the ability of astroglia to take up Pb from the culture medium and store it intracellularly, coupled with their resistance to overt Pb toxicity, it would be reasonable to postulate mechanisms of Pb tolerance in these cells. The bulk of experimental findings concerning low-level Pb exposure of cells supports two concepts: first, that Pb-induced damage is a continuous, cumulative process that begins at discrete molecular sites and progresses unless contained by defensive mechanisms within the cell; and second, that Pb infiltrates metabolic pathways normally used by essential metals. In the proposed study, three hypotheses will be tested, the first of which is that Pb enters astroglia via multiple mechanisms, including Ca2+ channels and anion exchangers. This hypothesis will be tested by spectrofluorometrically monitoring Pb2+ entry into cultured astroglia in the presence of ion channel agonists and blockers and anion transport blockers by the use of the Ca2+ fluorophore Fura-2, which binds-to Pb2+. Values for Km and Vmax that characterize the kinetics of Pb transport energy requirements for uptake will be determined, and the effects of competing metals and extracellular proteins will be assessed. The second hypothesis is that glutathione is a defense mechanism against intracellularly accumulated Pb. It will be tested by measuring cytosolic glutathione content in astroglia in culture by interactive laser cytometry as a function of Pb exposure and correlating cell injury to glutathione levels. In addition, Pb-induced cell injury will be quantified in glutathione-depleted and glutathione-enriched cells. The final hypothesis is that Pb infiltrates intracellular Ca stores, thereby disrupting Ca cycling and metabolic processes dependent on Ca2+ signaling. This hypothesis will be tested by characterizing an intracellular divalent cation pool in Pb-exposed astroglia that is mobilized by extracellular stimulation with ionomycin, identifying physiological agents that mimic the ionomycin effect, and quantitating immediate physiological consequences of divalent cation mobilization, including transient closing of gap junctions, activation of PKC, and activation of peptidases.

Agency
National Institute of Health (NIH)
Institute
National Institute of Environmental Health Sciences (NIEHS)
Type
Research Project (R01)
Project #
5R01ES005871-02
Application #
2154736
Study Section
Toxicology Subcommittee 2 (TOX)
Project Start
1993-04-01
Project End
1996-03-31
Budget Start
1994-04-01
Budget End
1995-03-31
Support Year
2
Fiscal Year
1994
Total Cost
Indirect Cost
Name
Texas A&M University
Department
Veterinary Sciences
Type
Schools of Veterinary Medicine
DUNS #
City
College Station
State
TX
Country
United States
Zip Code
77845
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Qian, Y; Mikeska, G; Harris, E D et al. (1999) Effect of lead exposure and accumulation on copper homeostasis in cultured C6 rat glioma cells. Toxicol Appl Pharmacol 158:41-9
Lindahl, L S; Bird, L; Legare, M E et al. (1999) Differential ability of astroglia and neuronal cells to accumulate lead: dependence on cell type and on degree of differentiation. Toxicol Sci 50:236-43