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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

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
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
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
0021-9258
7939-7949
Gray, Joshua P.
87bb062f-a135-4f9b-9b65-02ade9a7fece
Heck, Diane E.
03a96ab5-efa2-4374-a29f-a9ec70101fc0
Mishin, Vladimir
c29264bd-c415-4eed-906e-87fd95c961c5
Smith, Peter J.S.
003de469-9420-4f12-8f0e-8e8d76d28d6c
Hong, Jin-Yun
b71ccbd6-2d11-45a6-99ad-ac495e1b1c34
Thiruchelvam, Mona
73796476-5cb9-4f47-9b79-c86390a7a675
Cory-Slechta, Deborah A.
de0dbefe-7148-447f-888f-c45f197d7a3c
Laskin, Debra L.
898efc44-86dd-4de1-b381-fc04719018e4
Laskin, Jeffrey D.
8c4377b4-ba21-4811-8cb1-35904f35e1d5
Gray, Joshua P.
87bb062f-a135-4f9b-9b65-02ade9a7fece
Heck, Diane E.
03a96ab5-efa2-4374-a29f-a9ec70101fc0
Mishin, Vladimir
c29264bd-c415-4eed-906e-87fd95c961c5
Smith, Peter J.S.
003de469-9420-4f12-8f0e-8e8d76d28d6c
Hong, Jin-Yun
b71ccbd6-2d11-45a6-99ad-ac495e1b1c34
Thiruchelvam, Mona
73796476-5cb9-4f47-9b79-c86390a7a675
Cory-Slechta, Deborah A.
de0dbefe-7148-447f-888f-c45f197d7a3c
Laskin, Debra L.
898efc44-86dd-4de1-b381-fc04719018e4
Laskin, Jeffrey D.
8c4377b4-ba21-4811-8cb1-35904f35e1d5

Gray, Joshua P., Heck, Diane E., Mishin, Vladimir, Smith, Peter J.S., Hong, Jin-Yun, Thiruchelvam, Mona, Cory-Slechta, Deborah A., Laskin, Debra L. and Laskin, Jeffrey D. (2007) 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. The Journal of Biological Chemistry, 282 (11), 7939-7949. (doi:10.1074/jbc.M611817200). (PMID:17229725)

Record type: Article

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

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Published date: March 2007

Identifiers

Local EPrints ID: 188755
URI: http://eprints.soton.ac.uk/id/eprint/188755
ISSN: 0021-9258
PURE UUID: 8e033ddd-1eb5-4aa8-b29a-855ea4b59b0c
ORCID for Peter J.S. Smith: ORCID iD orcid.org/0000-0003-4400-6853

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Date deposited: 01 Jun 2011 10:17
Last modified: 26 Nov 2021 02:57

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Contributors

Author: Joshua P. Gray
Author: Diane E. Heck
Author: Vladimir Mishin
Author: Jin-Yun Hong
Author: Mona Thiruchelvam
Author: Deborah A. Cory-Slechta
Author: Debra L. Laskin
Author: Jeffrey D. Laskin

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