The mechanism of oxidative stress-induced inflammation in environmental- and metabolism-related human diseases is only partially understood. The long-term goal of this proposal is to apply insights and techniques discovered/developed in the present project to understand the molecular basis of environmental-lung-chronic inflammatory diseases. The specific hypothesis is: biomacromolecule-centered free radicals are part of the redox signaling triggered by lipopolysaccharide (LPS) during macrophage activation. This hypothesis is based on the following observations: 1) LPS induces reactive oxygen species (ROS) in cells;2) ROS can mobilize redox-active iron from lysosomes;3) iron binds to macromolecules and reacts in situ with ROS producing hydroxyl radicals;4) hydroxyl radicals abstract electron(s) from macromolecules producing free radicals.
The specific aims are designed to test our hypothesis using immuno-spin trapping (1ST) with the nitrone spin trap 5,5,-dimethyM-pyrroline A/-oxide (DMPO) and to improve our methods of detecting and characterizing those free radicals as their DMPO nitrone adduct-derivatives.
SPECIFIC AIM#1 : To determine the role of iron-catalyzed oxidations in triggering the redox signaling that produces LPS-induced activation of macrophages. To achieve this aim we will: a) determine the source and the type of ROS that triggers the redox signaling, b) determine whether the ROS induced by LPS mediates the release of iron from lysosomes to the cytoplasm, and c) study LPS-induced markers of iron-catalyzed oxidation of biomacromolecules during macrophage activation.
SPECIFIC AIM # 2: To optimize, validate, and apply 1ST with DMPO to characterize protein radicals produced during LPS-induced activation of macrophages. To achieve this aim we will: a) immuno(cyto)chemically analyze DMPO-protein nitrone adducts, b) prepare beads coated with anti-DMPO antibodies to immunopurify nitrone adducts, and c) characterize purified DMPO nitrone adducts by mass spectrometric analyses.
SPECIFIC AIM #3 : To optimize, validate, and apply 1ST with DMPO to.detect total mRNA radicals in LPS- induced activation of macrophages. To achieve this aim we will: a) detect DMPO-RNA nitrone adducts, b) develop and validate 1ST for detecting mRNA radicals, and c) apply 1ST to the detection of total mRNA radicals induced in LPS-activated macrophages and in the lungs of mice exposed to LPS.
New studies aimed at identifying novel targets and early markers of exposure will help us develop better diagnostic and therapeutic approaches for oxidative stress-induced inflammation-mediated human diseases.
