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Identification of an FAD-independent redox cycling activity for 9,10-phenanthrenequinonein mouse lung epithelial cells

TitleIdentification of an FAD-independent redox cycling activity for 9,10-phenanthrenequinonein mouse lung epithelial cells
Publication TypeConference Proceedings
Year of Conference2009
AuthorsYang S., Gray J.P., Mishin V., Wang Y., Heck D.E., Laskin D.L., Laskin J.D.
Conference NameProceedings of the Society of Toxicology 48th Annual Meeting and ToxExpo
Volume1
PaginationA307
Date PublishedMarch15-19, 2009
PublisherSociety of Toxicology
Conference LocationBaltimore, MD
Abstract

9,10-Phenanthraquinone (PQ) is a component of airborne particulate matter and can cause protein oxidation and cytotoxicity via a one or two-electron reduction. One elecron reduction of PQ results in a semiquinone radical; oxidation of the radical leads to the formation of reactive oxygen species including superoxide anion, hydrogen peroxide and hydroxyl radicals. In the lung, this can cause alveolar inflammation, epithelial cell damage and pulmonary fibrosis. Using mouse MLE-15 lung epithelial cells, we discovered a strong NADPH-dependent redox cycling activity in cytosolic cellular fractions. Purification and sequencing identified this material as sepiapterin reductase (SPR), an enzyme catalyzing the final step in the biosynthetic pathway of tetrahydrobiopterin. Human SPR was cloned and its enzymatic activities characterized. In addition to reducing sepiapterin, the enzyme readiily mediated PQ redox cycling as measured by hydrogen peroxide formation in the Amplex Red assay. Dicoumarol was found to be a noncompetitive inhibitor (IC50 = 200 nM) of sepiapterin reduction without inhibiting redox cycling. Similar selective inhibition of SPR was found with N-acetylserotonin (2.5 mM), indomethacin (8.1 mM), ethacrynic acid (22.9 mM), and rutin (24.0 mM) while PQ was a nonpcompetitive inhibitor of SPR activity. Sepiapterin reduction and redox cycling activity were not inhibited by diphenyleneiodonium, a finding consistent with the fact that SPR does not contain flavin cofactors. Taken together, our data identify a novel enzyme in lung epithelial cells that mediates chemical redox cycling. Redox cycling via SPR may be an important mechanism by which contaminants in airborne particulate matter redox cycle and initiate toxicity. Supported by CA100994, CA132624, CA093798, ES004738, ES005022, GM034310 and AR055073.

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