The ubiquity of exposure highlights the risk of Ni compounds to human health. Toxic and carcinogenic effects of Ni compounds in humans, experimental animals and cultured cells are well documented, but the cytoplasmic target and mechanisms of cell injury are not well defined. Ni2+ induces a dramatic perinuclear bundling of micro-tubules (MT), but little is known about how Ni2+ perturbs MT. That Ni2+ enhances the in vitro MT polymerization and the stability of MT polymers thus formed has led to the following hypothesis for Ni2+-inflicted cell injury: In vitro, Ni2+ binds to tubulin and enhances its polymerization through interactions with GTP and Mg2+. By prolonging dynamics in favor of assembly such that cytoplasmic MT become stabilized forming bundles through interactions with their associated proteins, and tubulin expression is enhanced, further favoring MT bundling. The perinuclear centrosome, which is the MT organizing center, may function as a """"""""depository"""""""" for cellular Ni2+, and thus facilitating the Ni2+ delivery to and interactions with nuclear components where Ni2+ genotoxic effects occur. Defects or perturbation in Mt structural organization (bundling) could translate into functional disorder or inability. The overall goals of this project is to test this hypothesis by achieving the following specific aims: 1. To characterize Ni2+ effects on in vitro tubulin polymerization focusing on Ni2+- tubulin binding, Ni2+/GTp/Mg2+ interactions and inhibition of GTP hydrolysis. 2. To correlate the physical/chemical form of Ni compounds with their MT injuring and cell transforming activities. 3. To characterize Ni2+ effects on 3T3 cells focusing on assessing MT stability, tubulin posttranslational modifications and tubulin mRNA expression. 4. To assess the role of cellular glutathione (GSH) and cytosolic and cytoskeletal protein sulfhydryl (P-SH_ in modulating Ni2+-induced MT bundling. 5. To assess the role of MT and actin filaments in phagocytosis of carcinogenic particulate Ni compounds and to test the hypothesis that the centrosome may function as a perinuclear """"""""depository"""""""" for Ni2+. 6. To assess the Mg2+ antagonism of Ni2+-induced MT bundling and cell injury. The outcome of the proposed research should help bring us closer to achieving the long-term objective, that is, to understand the mechanisms of cell injury up Ni2+ insult.
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