MECHANISMS OF CYTOSKELETAL INJURY BY HEAVY METALS

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Serious health problems can occur in workers exposed to metals in the workplace atmosphere. Chronic or acute exposure to fumes containing Hg, or Pb can result in anemia and neurological damage, while acute exposure to Cd, or As can result in emphysema. Long term exposure to As ultimately can lead to lung cancer, while long term exposure to Cd can result in severe renal damage. It is unclear which intracellular targets of these metals have a direct effect on the expression of their systemic toxicity. This project represents initial studies concerning the cytoskeleton as a target of intracellular damage by these metals. It is intended to establish a basis for future studies relating cytoskeletal damage to systemic toxicity. All these metals bind avidly to sulfhydryl groups. However, Cd, Hg, and Pb can also bind to and activate calmodulin. In contrast, As cannot activate calmodulin. Microtubules contain sulfhydryl groups that must remain reduced for assembly to occur, and to maintain the assembled state. In addition, microtubule dynamics are regulated by calcium, via activation of calmodulin. Thus Cd, Hg, and Pb may affect microtubules by sulfhydryl binding, or calmodulin binding while the effects of As would be due to sulfhydryl binding. Changes in the amount of total cellular protein and cytoskeletal protein sulfhydryl groups during metal exposure will be monitored with the DTNB colorimetric assay and the 3H-NEM labeling assay. Activation of calmodulin by Ca, Cd, Hg, and Pb will be studied by determining the effects of calmodulin inhibitors on the effects of Ca and other metals on microtubules. Calmodulin binding of metal ions will be determined by an enhancement of tubulin assembly in the presence of calmodulin by the chelating action of calmodulin. Finally, binding and activation of calmodulin is shown by the slower mobility of activated calmodulin in non-denaturing PAGE. Calmodulin dependent phosphorylation of cytoskeletal proteins is analyzed with SDS-PAGE and autoradiography.