Ozone is a major constituent of photochemical smog. The lung is the main site of ozone toxicity, and the ability of ozone inhalation to cause airways inflammation has been shown in both animal and human studies. However, the molecular mechanisms and sequence of pathophysiological reactions that occur in relevant lung cells and produce the inflammatory response to ozone are not well understood. The applicants have suggested that ozone s reactivity is so great that it reacts with the first species it contacts at the air/tissue boundary. The hypothesis is that ozone cannot penetrate far into the cells that line the airways, so many of the pulmonary effects and all of the extra-pulmonary effects of ozone must be caused by messenger species, lipid ozonation products (LOP). These LOP signal transduction species should mimic the effects of ozone itself and trigger the release of inflammatory mediators. If the theory is correct, then the identification of the LOP that are responsible for transmitting the message of ozone s damage further into the lung is crucial, since these compounds begin the process that ultimately leads to the physiological effects associated with ozone. Based on data already obtained in the applicants laboratories, specific classes of LOP can activate phospholipases and, in turn, trigger the release of endogenous mediators of inflammation. The overall goals of this proposal are to identify LOP that are produced during the ozonation of unsaturated fatty acids that are present in epithelial cell membranes. These LOP will be synthesized, isolated, and purified and tested for ability to activate important transmembrane signaling processes (phospholipases A2, C, and D) in an important target for ozone, (i.e., human airway epithelial cells).
The Specific Aims are: (i) synthesize, isolate, purify, and study the properties of a number of classes of LOP that preliminary data show or prediction is made that they will have biological activity; (ii) incorporate radiolabeled fatty acids into lipids in airway epithelial cells, expose the cells to ozone, and separate and identify the labeled LOP that are produced, in order to determine that the LOP are produced in these cells; (iii) preliminary data show that hydroxyhydroperoxide-containing LOP are more activating toward PLC in airway epithelial cells than are the aldehydic LOP that also are produced in the ozonation. Therefore, the lifetimes and structure-activity relation of this novel class of signal transduction species will be examined; (iv) the biological endpoints of specific LOP activation of phospholipases will be characterized.
