title: 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.
abstract: 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 microm) 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 microm), 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.
authors.affiliation: Department of Environmental and Occupational Medicine, University of Medicine and Dentistry of New Jersey-Robert Wood Johnson Medical School, Piscataway, New Jersey 08854, USA.
authors.forename: Joshua P
authors.initials: JP
authors.lastname: Gray
authors.forename: Diane E
authors.initials: DE
authors.lastname: Heck
authors.forename: Vladimir
authors.initials: V
authors.lastname: Mishin
authors.forename: Peter J S
authors.initials: PJ
authors.lastname: Smith
authors.forename: Jun-Yan
authors.initials: JY
authors.lastname: Hong
authors.forename: Mona
authors.initials: M
authors.lastname: Thiruchelvam
authors.forename: Deborah A
authors.initials: DA
authors.lastname: Cory-Slechta
authors.forename: Debra L
authors.initials: DL
authors.lastname: Laskin
authors.forename: Jeffrey D
authors.initials: JD
authors.lastname: Laskin
mesh_terms: D000818:Animals; D016466:CHO Cells; D006224:Cricetinae; D003412:Cricetulus; D003486:Cyanides / Q000494:pharmacology*; D004594:Electrophysiology; D004789:Enzyme Activation; D004847:Epithelial Cells / Q000187:drug effects* / Q000378:metabolism; D008168:Lung / Q000187:drug effects*; D051379:Mice; D009247:NADH, NADPH Oxidoreductases / Q000378:metabolism*; D010100:Oxygen / Q000378:metabolism; D010269:Paraquat / Q000494:pharmacology*; D051381:Rats; D012119:Respiration / Q000187:drug effects*; D013880:Thioredoxin-Disulfide Reductase / Q000378:metabolism / Q000502:physiology*
chemical_list: D003486:Cyanides; D009247:NADH, NADPH Oxidoreductases; D013880:Thioredoxin-Disulfide Reductase; D010269:Paraquat; D010100:Oxygen