Cells are exposed daily to endogenous oxidants resulting from normal metabolism. In addition lymphocytes, which function in the oxidative environment of inflammatory foci, are exposed to exogenous oxidants released by phagocytes. The level of intracellular glutathione (GSH), a major antioxidant, is critical in determining whether lymphocytes respond appropriately to activation signals. In addition, the importance of GSH in detoxification of xenobiotics and its involvement in drug resistance is well established. It is hypothesized that GSH synthesis in humans is regulated at many levels in a tissue-specific manner, that up-regulation of GSH synthesis occurs in response to GSH depletion or xenobiotics, and that down-regulation of GSH synthesis occurs when cellular GSH levels are adequate. We hypothesize that much of this regulation occurs at the level of gamma-glutamylcysteine synthetase (GCS), the rate-limiting enzyme involved in GSH synthesis. We further hypothesize that interindividual variation in GLCLC, the gene encoding the catalytic subunit of GCS, may be functionally significant and may influence the cellular capacity to produce GSH, especially under conditions of activation or stress. These studies focus on GCS and the genes which encode its subunits. Preliminary studies have determined that GLCLC is polymorphic.
The specific aims of the proposed grant are: 1) to determine the functional significance of interindividual variation in GLCLC (DNA cloning/sequencing, transfection systems for analyses of promoter activity, GCS activity), 2) to investigate transcriptional regulation of GLCLC in resting or activated human T cells exposed in vitro to environmental stressors (nuclear run-ons, primer extension, analyses of mRNA stability and mRNA translatability), 3) To analyze regulation of GCS subunits in human T cells in response to activation or stress (GCS activity, immunoprecipitations/Western blots, pulse-chase studies of protein stability, analysis of protein phosphorylation, cellular localization). The long-term goal of the proposed studies is to understand the regulation of GSH synthesis in human lymphoid cells and to determine whether certain alleles of GLCLC are associated with increased risk of disease subsequent to an individual''s exposure to environmental toxicants.
| von Stein, Richard T; Silver, Kristopher S; Soderlund, David M (2013) Indoxacarb, Metaflumizone, and Other Sodium Channel Inhibitor Insecticides: Mechanism and Site of Action on Mammalian Voltage-Gated Sodium Channels. Pestic Biochem Physiol 106:101-112 |
| Walsh, A C; Feulner, J A; Reilly, A (2001) Evidence for functionally significant polymorphism of human glutamate cysteine ligase catalytic subunit: association with glutathione levels and drug resistance in the National Cancer Institute tumor cell line panel. Toxicol Sci 61:218-23 |
