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J. Biol. Chem., Vol. 282, Issue 11, 7939-7949, March 16, 2007

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Paraquat Increases Cyanide-insensitive Respiration in Murine Lung Epithelial Cells by Activating an NAD(P)H:Paraquat Oxidoreductase

IDENTIFICATION OF THE ENZYME AS THIOREDOXIN REDUCTASE^*

Joshua P. Gray, Diane E. Heck, Vladimir Mishin, Peter J. S. Smith^¶, Jun-Yan Hong^||, Mona Thiruchelvam, Deborah A. Cory-Slechta, Debra L. Laskin, and Jeffrey D. Laskin¹

From the Department of Environmental and Occupational Medicine, University of Medicine and Dentistry of New Jersey-Robert Wood Johnson Medical School, Piscataway, New Jersey 08854, Department of Pharmacology and Toxicology, Rutgers University, Piscataway, New Jersey 08854, ^¶Biocurrents Research Center, Marine Biological Laboratory, Woods Hole, Massachusetts 02543, and ^||University of Medicine and Dentistry of New Jersey-School of Public Health, Piscataway, New Jersey 08854

Pulmonary fibrosis is one of the most severe consequences of exposure to paraquat, an herbicide that causes rapid alveolar inflammation and epithelial cell damage. Paraquat is known to induce toxicity in cells by stimulating oxygen utilization via redox cycling and the generation of reactive oxygen intermediates. However, the enzymatic activity mediating this reaction in lung cells is not completely understood. Using self-referencing microsensors, we measured the effects of paraquat on oxygen flux into murine lung epithelial cells. Paraquat (10–100 µM) was found to cause a 2–4-fold increase in cellular oxygen flux. The mitochondrial poisons cyanide, rotenone, and antimycin A prevented mitochondrial- but not paraquat-mediated oxygen flux into cells. In contrast, diphenyleneiodonium (10 µM), an NADPH oxidase inhibitor, blocked the effects of paraquat without altering mitochondrial respiration. NADPH oxidases, enzymes that are highly expressed in lung epithelial cells, utilize molecular oxygen to generate superoxide anion. We discovered that lung epithelial cells possess a distinct cytoplasmic diphenyleneiodonium-sensitive NAD(P)H:paraquat oxidoreductase. This enzyme utilizes oxygen, requires NADH or NADPH, and readily generates the reduced paraquat radical. Purification and sequence analysis identified this enzyme activity as thioredoxin reductase. Purified paraquat reductase from the cells contained thioredoxin reductase activity, and purified rat liver thioredoxin reductase or recombinant enzyme possessed paraquat reductase activity. Reactive oxygen intermediates and subsequent oxidative stress generated from this enzyme are likely to contribute to paraquat-induced lung toxicity.

Received for publication, December 26, 2006

^* This work was supported in part by National Institutes of Health Grants U54AR055073, ES006897, CA100994, CA093798, ES003647, ES010791, ES004738, GM034310, RR001395, and ES005022. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

¹ To whom correspondence should be addressed: Dept. of Environmental and Occupational Medicine, UMDNJ-Robert Wood Johnson Medical School, 170 Frelinghuysen Rd., Piscataway, NJ 08854. Tel.: 732-445-0176; Fax: 732-445-0119; E-mail: jlaskin{at}eohsi.rutgers.edu.

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