Investigation of endothelial cell injury and dysfunction induced by air pollutant benzo-a-pyrene-1,6-quinone: implications in chemical atherogenesis

UNCG Author/Contributor (non-UNCG co-authors, if there are any, appear on document)
Halley Shah (Creator)
The University of North Carolina at Greensboro (UNCG )
Web Site:
Zhenquan Jia

Abstract: Cardiovascular diseases (CVD) are the leading cause of death for men and women in the USA, and growing evidence has led to an increase in concern about the detrimental effect of airborne particulate matter (PM) on health and its relation to the development of CVD. Indeed, epidemiological studies have shown that there is a well-established association between PM and increased atherosclerosis morbidity and mortality. However, how PM induces atherosclerosis remains to be elucidated. PM is a heterogeneous mixture of incomplete combustion of organic matter and fossil fuels, which is made up of metals, aldehydes, polycyclic aromatic hydrocarbons (PAHs) including benzo-a-pyrene (BP), and a variety of quinones. BP is produced during incomplete combustion of organic matter such as coal, oil, garbage or other organic substances. BP-1,6-quinone (BP-1,6-Q) is derived from BP and is one of the important constituents of PM. It is generated through atmospheric transformations of BP by UV light or by the oxidation of BP via CYP450 enzymes within the cells. While endothelium has been suggested to be one of the well-known targets by air pollutants, the action of BP-1,6-Q in endothelial cells remains to be examined. We hypothesized that BP-1,6-Q could promote atherosclerosis through endothelial cell injury. The goal of this research project is to elucidate the effects and the underlying mechanism of BP-1,6-Q on endothelial injury using human EA.hy926 endothelial cells as a model system. The specific objectives of this study were; (1) to examine redox activity of BP-1,6-Q and the underlying molecular mechanisms involved, (2) to elucidate the role of cellular glutathione (GSH) in BP-1,6-Q- mediated cytotoxicity and cellular macromolecule damages, (3) to study the role of Phase II enzyme NADPH-quinone oxidoreductase-1 (NQO1) in BP-1,6-Q-mediated cytotoxicity and ROS production in human EA.hy926 endothelial cells. We first examined whether BP-1,6-Q at non-toxic concentrations (0.01-1 [lowercase Greek letter mu]M) could produce ROS in EA.hy926 endothelial cells as ROS is a key mediator of signaling pathways causing oxidative stress in the development of atherosclerosis. Using lucigenin- and luminol-derived chemiluminescence and 2’7’-Dichlorofluorescein diacetate (DCF-DA) flow cytometry assays, we have shown that BP-1,6-Q stimulated the production of reactive oxygen species (ROS) in a concentration-dependent manner. Furthermore, BP-1,6-Q-induced ROS was inhibited by rotenone (Rot) and antimycin A (AA), denoting the involvement of mitochondrial electron transport chain (METC) in redox cycling of BP-derived quinones in ROS overproduction. We have further demonstrated that BP-1,6-Q triggered endothelial-monocyte interaction and stimulated expression of vascular adhesion molecule-1 (VCAM-1). These results suggest that ROS production may partially mediate the inflammatory effect of BP-1,6-Q on endothelial inflammation. To further determine the mechanisms by which BP-1,6-Q produces ROS, we have determined the involvement of Phase II enzyme NQO1 in redox cycling of BP-1,6-Q. We have observed that inhibition of NQO1 showed a decrease in generation of BP-1,6-Q-mediated ROS, and augmentation of NQO1 resulted in a significant increase in BP-1,6-Q-induced ROS. Thus, our results reported for the first time that NQO1 plays a vital role in catalyzing redox activation of BP-1,6-Q to generate ROS in endothelial cells. In contrast, at higher concentrations (20-60 [lowercase Greek letter mu]M) BP-1,6-Q causes a significant decrease in cell viability and an increase in the necrotic type of cell death as measured by lactate dehydrogenase assay (LDH) and flow cytometric assays. We have also demonstrated that BP-1,6-Q imparts its toxicity by depleting cellular glutathione (GSH) and NQO1 resulting in cellular macromolecular damage in a concentration-dependent manner. Augmentation of cellular GSH and NQO1 showed significant protection against BP-1,6-Q-induced cell death. These results indicate that GSH and NQO1 might be first in the line of defense against BP-1,6-Q induced cytotoxicity. Interesting, our results further showed that NQO1-mediated ROS production by BP-1,6-Q is not associated with BP-1,6-Q induced cell injury suggesting that BP-1,6-Q can be toxic to endothelial cells by itself without any biotransformation to toxic metabolites. Taken together, this thesis study conducted using human EA.hy926 endothelial cells as a model system expanded our understanding of the possible involvement of mitochondria and cellular GSH and NQO1 in BP-1,6-Q mediated cytotoxicity as well its ROS production. These studies will contribute to our ability to assess the cardiovascular risk of human exposure to air pollutant to BP-1,6-Q.[This abstract has been edited to remove characters that will not display in this system. Please see the PDF for the full abstract.]

Additional Information

Language: English
Date: 2018
Air Particulate Matter, Atherosclerosis, Benzo-a-pyrene-1, 6-quinone, Endothelial cells, NQO1, Reactive oxygen species
Endothelial cells
Polycyclic aromatic hydrocarbons $x Physiological effect
Air $x Pollution
Cardiovascular system $x Diseases

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