Involvement of CYP 2C9 in Mediating the Proinflammatory Effects of Linoleic Acid in Vascular Endothelial Cells

AbstractObjective: Polyunsaturated fatty acids such as linoleic acid are well known dietary lipids that may be atherogenic by activating vascular endothelial cells. In the liver, fatty acids can be metabolized by cytochrome P450 (CYP) enzymes, but little is known about the role of these enzymes in the vascular endothelium. CYP 2C9 is involved in linoleic acid epoxygenation, and the major product of this reaction is leukotoxin (LTX). We investigated the role of CYP-mediated mechanisms of linoleic acid metabolism in endothelial cell activation by examining the effects of linoleic acid or its oxidized metabolites such as LTX and leukotoxin diol (LTD).Methods: The effect of linoleic acid on CYP 2C9 gene expression was studied by RT-PCR. Oxidative stress was monitored by measuring DCF fluorescence and intracellular glutathione levels, and electrophoretic mobility shift assay was carried out to study the activation of oxidative stress sensitive transcription factors. Analysis of oxidized lipids was carried out by liquid chromatography/mass spectrometry.Results: Linoleic acid treatment for six hours increased the expression of CYP 2C9 in endothelial cells. Linoleic acid-mediated increase in oxidative stress and activation of AP-1 were blocked by sulfaphenazole, a specific inhibitor of CYP 2C9. The linoleic acid metabolites LTX and LTD increased oxidative stress and activation of transcription factors only at high concentrations.Conclusion: Our data show that CYP 2C9 plays a key role in linoleic acid-induced oxidative stress and subsequent proinflammatory events in vascular endothelial cells by possibly causing superoxide generation through uncoupling processes.Keywordslinoleic acidCYP 2C9leukotoxinleukotoxin dioloxidative stressKeywordsCYP = cytochrome P450ROS = reactive oxygen speciesLTX = leukotoxinLTD = leukotoxin diolLA = linoleic acidH2DCF-DA = Dichlorodihydrofluorescein diacetateMnTMPyP = [Mn (III) tetrakis (1-methyl-4-pyridyl) porphyrin pentachloride]AP-1 = activator protein 1NF-κB = nuclear factor κB This study was supported in part by grants from NIEHS/NIH (Superfund Basic Research Program Grants P42 ES07380 and P42 ES04699), USDA/NRI (2001-01054 and 00-35200-9101), DOD (DAMD17-99-9247), NIEHS Center for Children’s Environmental Health & Disease Prevention P01 ES11269, the M.I.N.D. Institute Research Program, and the Kentucky Agricultural Experimental Station.NotesPresented as a ACN New Investigator Award lecture by Dr. Saraswathi Viswanathan at the 43rd Annual ACN Meeting in San Antonio, TX.