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

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 M) 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 M), 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.Exposure of humans and animals to toxic doses of paraquat (1,1Ј-dimethyl-4,4Ј-bipyridylium) is known to damage the lung leading to pulmonary edema and hypertension, acute respiratory distress syndrome, and progressive lung fibrosis (1). In target cells paraquat undergoes redox cycling which may contribute to its toxic actions. Several mammalian NADPH oxidases have been identified as potential inducers of paraquat redox cycling including cytochrome P450 reductase and nitric-oxide synthase (2, 3). These enzymes generate a reduced paraquat radical that can act as an electron donor (4) (see Reaction 1). Reacting rapidly with molecular oxygen, the paraquat radical recycles back to paraquat and in the process forms highly toxic oxidants including superoxide anion, hydrogen peroxide, hydroxyl radicals, and in the presence of nitric oxide, peroxynitrite (5-7). Cellular damage generated by these oxidants, including lipid peroxidation, may be important in paraquatinduced lung damage (8, 9).Redox cycling reactions are known to consume significant quantities of oxygen (10 -12). In cells, this has the potential to reduce levels of oxygen available for metabolic processes resulting in oxidative stress and toxicity (13). Previous studies have demonstrated that paraquat can stimulate oxygen uptake by microsomes from rat and rabbit liver and rabbit lung as...

Cytotoxicity of paraquat in microglial cells: Involvement of PKCδ- and ERK1/2-dependent NADPH oxidase

“…In a recent study with lung cells, paraquat was shown to cause a 2-4 fold increase in cellular oxygen consumption, which was neither due to NADPH oxidase nor mitochondrial respiration (Gray et al, 2007). This study further indicated the presence of a specific NADPH:paraquat oxidoreductase in the lung cells for initiating the redox mechanism (Gray et al, 2007). Whether similar mechanism exists in microglial cells is not known and remains to be investigated.…”

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Sepiapterin Reductase Mediates Chemical Redox Cycling in Lung Epithelial Cells

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