PROJECT SUMMARY Dietary consumption of ω-3 and ω-6 fatty acids have been linked to cardiovascular health benefits in humans. The central hypothesis is that the cardiovascular physiological effects of ω-3 and ω-6 fatty acids are partly mediated by the synthesis of eicosanoids via the epoxygenase (EPOX) pathway. Herein we perform biochemical studies of some of the key enzymes in these pathways. CYP2J2 is an enzyme in the EPOX pathway that is highly expressed in the cardiovascular system in the aortic epithelium and cardiomyocytes. CYP2J2's primary effects are facilitated via epoxidation of ω-3 and ω-6 fatty acids into epoxides that exert potent anti- inflammatory, vasodilatory and pro-angiogenic effects. CYP2J2 is also implicated in cardiotoxicity of drugs. Additionally, CYP2J2 is also a membrane bound protein and exhibit unique biochemical mechanisms that are poorly characterized and are the primary focus of the current proposal. Our first goal is to understand allosteric modulation of CYP2J2 epoxygenase activity by ω-3 and ω-6 fatty acids and selected cardiotoxic drugs (doxorubicin, ebastine and terfenadine). Our second goal is to examine the metabolism of ω-3 and ω-6 fatty acid derived endocannabinoids by CYP2J2. It is predicted that similar to ω-6 endocannabinoids, the ω-3 endocannabinoids are substrates for the EPOX enzymes producing novel bioactive epoxide mediators. The third goal is to examine how the composition of membranes effect CYP2J2 activity. We use several novel approaches that includes detection of lipid mediators with mass spectrometry, innovative methodologies such as Nanodiscs to solubilize CYP2J2 and provide membrane bilayer environment. We also introduce novel concepts of lipid-drug heterotropic interactions influencing the formation of the products of these enzymes. The long-term goal of this work is to understand the interplay of the formation of the eicosanoids from dietary fatty acids.