The proposed project aims to determine if Cd exposure causes a redistribution of intrinsic stores of Cu within the cell. It is hypothesized that metallothionein (MT), induced by exposure to Cd, acts as a thermodynamic and kinetic trap for metals such as Cu which show a higher affinity and binding constant for the protein. The sequestration of Cu from intracellular proteins and enzymes requiring its presence either allosterically or catalytically for normal functioning may result ultimately in cellular pathologies. Consequently, the toxicological effects noted with Cd may arise from cellular deficiencies in Cu rather than excesses of Cd. Preliminary data from two invertebrate phyla are provided in support of this hypothesis. Experiments are proposed to evaluate this proposition further both in vitro using cultured mammalian cells and in vivo using the invertebrate mollusc Littorina littorea as a model organism. The five specific aims of the study are to determine whether : i) MT induction in response to Cd exposure results in a concomitant increase in the cellular/organismal burden of Cu; ii) MT induction alters the subcellular distribution of Cu, Zn and other physiologically important metals; iii) the observed changes in Cu are due to a redistribution of intrinsic stores or the accumulation of extrinsic Cu from the surrounding media, iv) there is impaired functioning of Cu containing enzymes (eg. cytochrome C oxidase activity) as a result of Cd exposure: v) the cytotoxic effects of Cd can be remedied by administering Cu; Two novel applications of inductively coupled plasma mass spectroscopy (ICP-MS) have been developed to study these investigatory questions. Firstly, an ICP-MS has been coupled directly to a high performance liquid chromatography system to allow the simultaneous separation and elemental analysis of MT and other cytosolic metal binding ligands. Secondly, protocols using stable isotopic 63Cu and 65 Cu have been developed to permit long term kinetic studies of Cu metabolism to be undertaken. The continued refinement and development of techniques with these capabilities will undoubtedly facilitate the study of Menke's and Wilson's disease and other genetic disorders which result from abnormal Cu metabolism. Finally, in addition to the studies of MT, the role of intracellular, mineralized orth/pyrophosphate containing inclusions in the metabolism of Cd will also be studies. Recent evidence has shown that these deposits may be an important site for the sequestration and deposition of accumulated Cd. In combination, these studies will provide important information on the competitive interactions between essential and non-essential metals for intracellular metal-binding ligands. An appreciation of these types of interaction are important if we are to understand the mechanisms by which metals exert their toxicological action. The proposed research program is particularly suitable for both graduate and undergraduate minority student involvement and provides broad exposure to a number of modern techniques that are commonly employed in the biomedical sciences including aseptic and metal-free clean technique, cell culture, electron microscopy, plasma spectroscopy and protein separation and purification. This project therefore provides an excellent opportunity for students to be exposed to the biomedical research experience.
