Studies are proposed in this application to evaluate the mammalian stress response to heavy metal injury and its role in the adaptation to adverse conditions posed by environmental chemicals. Our focus will be the induction of the endoplasmic reticulum (ER) chaperone glucose-regulated protein 78 (GRP78) in mammalian cells by lead (Pb) acetate and HgCl2. These metals in combination with other contaminants present at Superfund sites pose serious risks to human and animal health. Because the nature of toxicological interactions in complex chemical mixture remains poorly defined, experiments will be conducted to examine the molecular basis of the adaptive response to environmental injury. Specifically, we will test two hypothesis: that GRP78 is regulated transcriptionally following metal-induced alterations in signal transduction, and that this response affords cytoprotection against subsequent chemical insult. Gene regulatory mechanisms involved in cytoprotection will be examined in SCC-1, a rat mesangial clonal cell line, and C6, a rat glioma cell line. Treatment regimens will model toxicologically relevant exposures in humans by use of repeated dosing with Pb acetate and HgCl2 at sub-cytotoxic concentration over a period of days. In the first aim, we will determine if metal challenge perturbs Ca2+ homeostasis, redox status and/or protein phosphorylation, leading to activation of GRP78. The contribution of transcriptional and post- transcriptional mechanisms of gene induction, and the role of xenobiotic- regulated cis-acting elements within the 5' regulatory region of the gene will be examined. The cytoprotective effects of increased amounts of GRP78. The contribution of transcriptional and post-transcriptional mechanisms to gene induction, and the role of xenobiotic-regulated cis- acting elements within the 5' regulatory region of the gene will be examined. The cytoprotective effects of increased amounts of GRP78 in metal-treated cells, as evidenced by measurements of Ca2+ homeostasis, misfolded protein accumulation, cytokine release, and metal binding will be examined. Cellular tolerance to subsequent injury by chemical mixtures present at Superfund sites will be monitored to define relevant toxicologic interactions. Our approach will be tied to the imaging, field services and analytical cores, as well as Projects 2, 5 and 6 of this Program. Together the proposed studies will provide information about critical chemico-biologic interactions at Superfund sites.
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