Ozone, lipid-protein adducts, and biological effects

DESCRIPTION (provided by applicant): Ozone is one of the most commonly encountered environmental pollutants and human exposures to increased levels of this reactive molecule are clearly linked to proinflammatory responses and exacerbation of respiratory illness. Unsaturated lipids are particularly vulnerable to ozone and a number of electrophilic aldehydes and epoxides are known as primary products of lipid ozone exposure. Cholesterol, for example, reacts readily with ozone and gives oxysterol products that result from conversion of the ring- B double bond of the sterol to reactive carbonyl and epoxide functionality. Endogenously formed oxysterols are well-known ligands for the liver-X-receptor (LXR), which regulates the expression of genes involved in cholesterol homeostasis, fatty acid synthesis, and reverse cholesterol transport. More recent studies also implicate LXR in having potent anti-inflammatory and immune regulatory function. In contrast to endogenously formed oxysterols, some of the ozone-derived oxysterols inhibit LXR function, yet the role of this mechanism in ozone pathogenesis is known. Ozone is known to modify cellular function and activate epithelial cells but the biochemical mechanisms of these processes have yet to be defined. Ozone-derived oxysterols are electrophiles that react with common nucleophilic residues present in proteins, forming stable adducts. We contend that the known chemical reactivity of ozone, its presumed exposure to lipids in the airway, the oxidative stress that results from these exposures, and the association of human diseases with environmental insults calls for coordinated studies aimed at discovering the fundamental chemical and biological events linking lipids, environmental exposures and the pathophysiological response. Ozone-derived oxysterols are a common theme in the proposed research and we outline strategies here that are designed to: 1. Provide chemically pure ozone-derived oxysterols to assess the effect of these compounds and their metabolites on the function of epithelial cells, with specific focus on the LXR signaling pathway. 2. Develop methods based on click chemistry and synthetic alkynyl sterol analogs that permit the isolation and identification of oxysterol-protein adducts and define the oxysterol adduction proteome in epithelial cells and macrophages. 3. Utilize synthetic sterol and oxysterol analogs to track and identify lipids in various epithelial cellular compartments. 4. Develop assays for biomarkers of lipid-protein adduction based on our cellular studies. The laboratories at Vanderbilt University and University of North Carolina at Chapel Hill provide a unique combination of expertise for the study of an important environmental problem. This expertise includes skills in chemical synthesis, isolation and characterization coupled with experience studying adverse health effects induced upon exposure to air pollutants and the cellular mechanisms mediating these responses.