CELLULAR INTERACTIONS OF LEAD, CALCIUM, AND ZINC

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Lead toxicity continues to be one of the major preventable health hazards of children in this country. Recent estimates by the U.S. Department of Public Health indicate that between three and four million children are at risk for adverse health effects from lead (ATSDR, 1988). Complex interactions between lead and essential elements, primarily calcium, iron, zinc and copper are reported in the clinical and experimental literature. The mechanisms and toxicological significance of these interactions is not clearly understood. The objective of this application is to elucidate the effects of lead on hormone-signal transduction processes. This application for competitive renewal continues investigation into the cell biology of Pb2+-Ca2+ interactions, with new initiatives to elucidate the molecular site of action of Pb2+ on regulation of the osteocalcin gene. Together these experiments test the hypothesis that lead toxicity is expressed via a perturbation of the calcium messenger system and zinc-mediated cell functions. The specific aims of this proposal are to: 1. Investigate the effects of lead on the mobilization of [Ca2+]i by the second messengers myo-inositol 1,4,5-tris- phosphate or arachidonic acid in permeabilized ROS cells. Experiments will be conducted using 45Ca and ion selective electrodes to gain insight into the action of Pb2+ on second messenger function. 2. Investigate the ability of the second messengers, myo- inositol, 1,4,5-tris-phosphate and arachidonic acid to mobilize Pb2+ from intracellular stores in ROS cells. Experiments will be conducted using 210Pb to gain insight into the dynamic state of Pb+ during signal-transduction processes. 3. Characterize the effects of Pb2+ on calmodulin and protein kinase C-mediated functions. Broad spectrum effects of lead on calmodulin and protein kinase C will be established by 2-d electrophoretic analysis of protein phosphorylation. The a more specific action of Pb2+ on a protein kinase C-mediated function will be established by monitoring the hormonal regulation of ornithine decarboxylase. 4. Elucidate the mechanism of action by which inhibits the induction of osteocalcin by 1,25-dihydroxyvitamin D3 in osteoblastic bone cells focusing on the Zn2+-mediated aspects of this process. Experiments will be conducted to (a) determine the displacement of Zn2+ and binding of Pb2+ at the zinc-fingers of the 1,25(OH)2D3 receptor, (b) investigate the effects of lead on receptor translocation to the nucleus, (c) receptor binding to DNA, and finally (d) transcription and translation of the osteocalcin gene. The concepts and results developed by this project should contribute directly to understanding the skeletal metabolism of lead, the skeleton as a target of lead toxicity, and the physiological factors which may mobilize skeletal lead. The cellular and molecular concepts defining the actions of lead on hormone signal-transduction processes should also be broadly applicable to understanding the effects of lead on growth, hypertensin, and behavior and learning deficits.