Altering nitric oxide bioavailability and lipid profiles in endothelial cells by polycyclic aromatic hydrocarbons in particulate matter

ALTERING NITRIC OXIDE BIOAVAILABILITY AND LIPID PROFILES IN ENDOTHELIAL CELLS BY POLYCYCLIC AROMATIC HYDROCARBONS IN PARTICULATE MATTER Liang Yu Old Dominion University, 2011 Co-Directors: Dr. R. James Swanson Dr. H. Anna Jeng Epidemiologic studies have demonstrated a significant association between exposure to particulate matter (PM) and atherosclerosis. Polycyclic aromatic hydrocarbons (PAHs) present in particulate matter, are well known to induce oxidative stress and lipid peroxidation via generation of reactive oxygen species (ROS). Lipid peroxidation involves regulating endothelial nitric oxide synthase via inhibition of its activity, and as a result, mediates dilation of coronary arterioles is involved with the pathogenesis of atherosclerosis. However, data on assessment of oxidized lipid formation is limited due to low resolution of mass spectrometer methods. Taking the advantage of a Fourier Transform Ion Cyclotron Resonance Mass Spectrometer (FT-ICRMS) with 12 Tesla at Old Dominion University, this study assessed how lipid peroxidation induced by PAHs altered the lipid profile and nitric oxide level in human endothelial cells. Human coronary artery endothelial cells (HCAEC) were exposed to PAHs with various doses and treatment duration times. After exposure, the level of ROS was measured using the fluorometric method with a flow cytometer. Lipid peroxidation was assessed based on the formation of malondialdehyde. Nitric oxide synthase was calculated using a cell-permeable diacetate that reacts with NO to form a fluorescent triazolofluorescein. Then, lipids of HCAEC were extracted by chloroform and methanol and analyzed using 12T FT-ICR-MS to separate peaks. After PAH exposure, cell morphology noticeably changed, granularity increased, and viability decreased after the short term treatment. The ROS level, expressed as fluorescent intensity readings from a flow cytometer, significantly increased along with the malondialdehyde levels. The analysis of spectrum data from FT-ICR-MS showed changes of the cellular lipid profile in the exposed groups as compared to the control. We found no significant impacts of oxidative stress on nitric oxide bioavailability in endothelial cells. However, direct exposure of HCAEC to PAHs increased lipid oxidization. These data imply that PAHs may mediate nitric oxide synthase activity through lipid peroxidation by a mechanism yet to be elucidated. These studies demonstrate one potential means by which oxidative stress can induce endothelial cell damage and lipid peroxidation during atherosclerosis for a short term exposure. In conclusion, the oxidation pathway induced by PAHs contributed to endothelial cell damage. This study identified lipids and developed novel data analytic approaches that are applicable in revealing oxidative lipid formation induced by PAHs. In addition, the technology enables us to identify specifically oxidized phospholipids that could serve as biomarkers in assessing PAH-induced endothelial dysfunction and the underlying mechanism. Copyright, 2011, by Liang Yu, H. Anna Jeng and Dr. R. James Swanson, All Rights Reserved.