Redox cycling and increased oxygen utilization contribute to diquat-induced oxidative stress and cytotoxicity in Chinese hamster ovary cells overexpressing NADPH-cytochrome P450 reductase
Redox cycling and increased oxygen utilization contribute to diquat-induced oxidative stress and cytotoxicity in Chinese hamster ovary cells overexpressing NADPH-cytochrome P450 reductase
Diquat and paraquat are nonspecific defoliants that induce toxicity in many organs including the lung, liver, kidney, and brain. This toxicity is thought to be due to the generation of reactive oxygen species (ROS). An important pathway leading to ROS production by these compounds is redox cycling. In this study, diquat and paraquat redox cycling was characterized using human recombinant NADPH-cytochrome P450 reductase, rat liver microsomes, and Chinese hamster ovary (CHO) cells constructed to overexpress cytochrome P450 reductase (CHO-OR) and wild-type control cells (CHO-WT). In redox cycling assays with recombinant cytochrome P450 reductase and microsomes, diquat was 10-40 times more effective at generating ROS compared to paraquat (K(M)=1.0 and 44.2?M, respectively, for H(2)O(2) generation by diquat and paraquat using recombinant enzyme, and 15.1 and 178.5?M, respectively for microsomes). In contrast, at saturating concentrations, these compounds showed similar redox cycling activity (V(max)?6.0nmol H(2)O(2)/min/mg protein) for recombinant enzyme and microsomes. Diquat and paraquat also redox cycle in CHO cells. Significantly more activity was evident in CHO-OR cells than in CHO-WT cells. Diquat redox cycling in CHO cells was associated with marked increases in protein carbonyl formation, a marker of protein oxidation, as well as cellular oxygen consumption, measured using oxygen microsensors; greater activity was detected in CHO-OR cells than in CHO-WT cells. These data demonstrate that ROS formation during diquat redox cycling can generate oxidative stress. Enhanced oxygen utilization during redox cycling may reduce intracellular oxygen available for metabolic reactions and contribute to toxicity
874-882
Fussell, Karma C.
0e577047-121f-4ee0-a022-89ac2edea454
Udasin, Ronald G.
01264bc8-50e2-45c4-ab8d-691acdea43a3
Gray, Joshua P.
87bb062f-a135-4f9b-9b65-02ade9a7fece
Mishin, Vladimir
c29264bd-c415-4eed-906e-87fd95c961c5
Smith, Peter J.S.
003de469-9420-4f12-8f0e-8e8d76d28d6c
Heck, Diane E.
03a96ab5-efa2-4374-a29f-a9ec70101fc0
Laskin, Jeffrey D.
8c4377b4-ba21-4811-8cb1-35904f35e1d5
April 2011
Fussell, Karma C.
0e577047-121f-4ee0-a022-89ac2edea454
Udasin, Ronald G.
01264bc8-50e2-45c4-ab8d-691acdea43a3
Gray, Joshua P.
87bb062f-a135-4f9b-9b65-02ade9a7fece
Mishin, Vladimir
c29264bd-c415-4eed-906e-87fd95c961c5
Smith, Peter J.S.
003de469-9420-4f12-8f0e-8e8d76d28d6c
Heck, Diane E.
03a96ab5-efa2-4374-a29f-a9ec70101fc0
Laskin, Jeffrey D.
8c4377b4-ba21-4811-8cb1-35904f35e1d5
Fussell, Karma C., Udasin, Ronald G., Gray, Joshua P., Mishin, Vladimir, Smith, Peter J.S., Heck, Diane E. and Laskin, Jeffrey D.
(2011)
Redox cycling and increased oxygen utilization contribute to diquat-induced oxidative stress and cytotoxicity in Chinese hamster ovary cells overexpressing NADPH-cytochrome P450 reductase.
Free Radical Biology and Medicine, 50 (7), .
(doi:10.1016/j.freeradbiomed.2010.12.035).
(PMID:21215309)
Abstract
Diquat and paraquat are nonspecific defoliants that induce toxicity in many organs including the lung, liver, kidney, and brain. This toxicity is thought to be due to the generation of reactive oxygen species (ROS). An important pathway leading to ROS production by these compounds is redox cycling. In this study, diquat and paraquat redox cycling was characterized using human recombinant NADPH-cytochrome P450 reductase, rat liver microsomes, and Chinese hamster ovary (CHO) cells constructed to overexpress cytochrome P450 reductase (CHO-OR) and wild-type control cells (CHO-WT). In redox cycling assays with recombinant cytochrome P450 reductase and microsomes, diquat was 10-40 times more effective at generating ROS compared to paraquat (K(M)=1.0 and 44.2?M, respectively, for H(2)O(2) generation by diquat and paraquat using recombinant enzyme, and 15.1 and 178.5?M, respectively for microsomes). In contrast, at saturating concentrations, these compounds showed similar redox cycling activity (V(max)?6.0nmol H(2)O(2)/min/mg protein) for recombinant enzyme and microsomes. Diquat and paraquat also redox cycle in CHO cells. Significantly more activity was evident in CHO-OR cells than in CHO-WT cells. Diquat redox cycling in CHO cells was associated with marked increases in protein carbonyl formation, a marker of protein oxidation, as well as cellular oxygen consumption, measured using oxygen microsensors; greater activity was detected in CHO-OR cells than in CHO-WT cells. These data demonstrate that ROS formation during diquat redox cycling can generate oxidative stress. Enhanced oxygen utilization during redox cycling may reduce intracellular oxygen available for metabolic reactions and contribute to toxicity
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Published date: April 2011
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Local EPrints ID: 190493
URI: http://eprints.soton.ac.uk/id/eprint/190493
ISSN: 0891-5849
PURE UUID: b095937c-2a10-4493-a2e4-5a2b49eb3541
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Date deposited: 13 Jun 2011 10:44
Last modified: 15 Mar 2024 03:38
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Author:
Karma C. Fussell
Author:
Ronald G. Udasin
Author:
Joshua P. Gray
Author:
Vladimir Mishin
Author:
Diane E. Heck
Author:
Jeffrey D. Laskin
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