Title: 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 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. 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. Exposure of humans and animals to toxic doses of paraquat (1,1′-dimethyl-4,4′-bipyridylium) is known to damage the lung leading to pulmonary edema and hypertension, acute respiratory distress syndrome, and progressive lung fibrosis (1Kimbrough R.D. Gaines T.B. Toxicol. Appl. Pharmacol. 1970; 17: 679-690Crossref PubMed Scopus (99) Google Scholar). 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 (2Day B.J. Patel M. Calavetta L. Chang L.Y. Stamler J.S. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 12760-12765Crossref PubMed Scopus (168) Google Scholar, 3Clejan L.A. Cederbaum A.I. Biochem. J. 1993; 295: 781-786Crossref PubMed Scopus (17) Google Scholar). These enzymes generate a reduced paraquat radical that can act as an electron donor (4Michaelis L. Hill E.S. J. Am. Chem. Soc. 1933; 55: 1481-1494Crossref Scopus (104) Google Scholar) (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 (5Rashba-Step J. Cederbaum A.I. Mol. Pharmacol. 1994; 45: 150-157PubMed Google Scholar, 6Bonneh-Barkay D. Reaney S.H. Langston W.J. Di Monte D.A. Mol. Brain Res. 2005; 134: 52-56Crossref PubMed Scopus (135) Google Scholar, 7Dicker E. Cederbaum A.I. Biochem. Pharmacol. 1991; 42: 529-535Crossref PubMed Scopus (47) Google Scholar). Cellular damage generated by these oxidants, including lipid peroxidation, may be important in paraquat-induced lung damage (8Karakashev P. Petrov L. Alexandrova A. Neoplasma. 2000; 47: 122-124PubMed Google Scholar, 9Schweich M.D. Lison D. Lauwerys R. Biochem. Pharmacol. 1994; 47: 1395-1400Crossref PubMed Scopus (15) Google Scholar). Redox cycling reactions are known to consume significant quantities of oxygen (10McCord J.M. Am. J. Med. 2000; 108: 652-659Abstract Full Text Full Text PDF PubMed Scopus (1078) Google Scholar, 11Mason R.P. Environ. Health Perspect. 1990; 87: 237-243Crossref PubMed Scopus (61) Google Scholar, 12Kappus H. Biochem. Pharmacol. 1986; 35: 1-6Crossref PubMed Scopus (352) Google Scholar). In cells, this has the potential to reduce levels of oxygen available for metabolic processes resulting in oxidative stress and toxicity (13Prabhakar N.R. Kumar G.K. Biol. Chem. 2004; 385: 217-221Crossref PubMed Scopus (103) Google Scholar). Previous studies have demonstrated that paraquat can stimulate oxygen uptake by microsomes from rat and rabbit liver and rabbit lung as well as rabbit lung slices, alveolar macrophages, and lung fibroblasts and in Escherichia coli (14Gage J.C. Biochem. J. 1968; 109: 757-761Crossref PubMed Scopus (157) Google Scholar, 15Rossouw D.J. Engelbrecht F.M. S. Afr. Med. 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Based on these findings, it has been suggested that paraquat functions by diverting electron flow from cyanide-sensitive respiration (18Hassan H.M. Fridovich I. J. Biol. Chem. 1977; 252: 7667-7672Abstract Full Text PDF PubMed Google Scholar). In the present studies we report that paraquat treatment of murine lung epithelial cells markedly increases the flux of oxygen into cells in a process that is independent of cyanide-sensitive respiration. These results are consistent with enhanced oxygen utilization during paraquat redox cycling (22Rossouw D.J. Engelbrecht F.M. S. Afr. Med. J. 1978; 54: 199-201PubMed Google Scholar). We also report that redox cycling appears to be initiated by a one-electron reduction of the herbicide by a specific NAD(P)H oxidase. This enzymatic activity was purified and shown to generate paraquat radical and to mediate production of ROI. 2The abbreviations used are: ROI, reactive oxygen intermediates; DPI, diphenyleneiodonium; NAME, nitroarginine methyl ester; MLE-15, murine lung epithelial cells; DTNB, 5,5′-dithiobis(2-nitrobenzoic acid); ATM, sodium aurothiomalate; CHO, Chinese hamster ovary; HPLC, high performance liquid chromatography. Thioredoxin reductase, an important antioxidant enzyme known to reduce thioredoxin as well as a number of other oxidants, was identified as the active enzyme (23Spyrou G. Holmgren A. Biochem. Biophys. Res. Commun. 1996; 220: 42-46Crossref PubMed Scopus (28) Google Scholar, 24Karimpour S. Lou J. Lin L.L. Rene L.M. Lagunas L. Ma X. Karra S. Bradbury C.M. Markovina S. Goswami P.C. Spitz D.R. Hirota K. Kalvakolanu D.V. Yodoi J. Gius D. Oncogene. 2002; 21: 6317-6327Crossref PubMed Scopus (106) Google Scholar, 25Holmgren A. Lyckeborg C. Proc. Natl. Acad. Sci. U. S. 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Rat cytochrome P450 reductase (Supersomes™, catalog no. 456514) was from BD Biosciences. Polyclonal antibodies to rat cytochrome P450 reductase (whole rabbit serum, catalog no. OSA-300) were from Stressgen (Victoria, BC). Paraquat, NADPH, NADH, horseradish peroxidase, 2′,5′-ADP-agarose, and all other reagents were purchased from Sigma-Aldrich. The Superose 12 HR 10/30 size exclusion column was obtained from Amersham Biosciences. Rat liver thioredoxin reductase was obtained from Cayman Chemical (Ann Arbor, MI). Cells and Treatments—MLE-15 murine lung epithelial cells were kindly provided by Dr. Jacob Finkelstein (University of Rochester) (30Barrett E.G. Johnston C. Oberdorster G. Finkelstein J.N. Am. J. Physiol. 1998; 275: L1110-L1119PubMed Google Scholar). Parental CHO cells were obtained from the American Type Culture Collection (Manassas, VA). The preparation of CHO cells expressing cytochrome P450 reductase or control empty vector has been described previously (31Han J.F. Wang S.L. He X.Y. Liu C.Y. Hong J.Y. Toxicol. Sci. 2006; 91: 42-48Crossref PubMed Scopus (41) Google Scholar). Cells were maintained in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum, penicillin (100 units/ml), and streptomycin (100 μg/ml) at 37 °C in 5% CO2 in a humidified incubator. Tissue culture reagents were from Invitrogen. For recordings, cells were plated at varying densities (0.05–0.5 × 105 cells) onto 35-mm plastic Petri dishes in phenol red-free Dulbecco's modified Eagle's medium with 10% fetal calf serum. At low density cells grow individually, whereas at higher densities cells grow as confluent monolayers. Recordings were performed in phosphate-buffered saline. Paraquat and other reagents were added sequentially directly to the medium. Recordings were suspended during the 10–15 s required to apply the drugs. Time was recorded continuously during the addition of reagents. Self-referencing Electrodes—The preparation and characterization of the oxygen microsensors has been described previously (32Jung S.K. Trimarchi J.R. Sanger R.H. Smith P.J. Anal. Chem. 2001; 73: 3759-3767Crossref PubMed Scopus (29) Google Scholar, 33Land S.C. Porterfield D.M. Sanger R.H. Smith P.J. J. Exp. Biol. 1999; 202: 211-218Crossref PubMed Google Scholar, 34Porterfield D.M. Corkey R.F. Sanger R.H. Tornheim K. Smith P.J. Corkey B.E. Diabetes. 2000; 49: 1511-1516Crossref PubMed Scopus (52) Google Scholar, 35Porterfield D.M. Laskin J.D. Jung S.K. Malchow R.P. Billack B. Smith P.J. Heck D.E. Am. J. Physiol. Lung Cell. Mol. Physiol. 2001; 281: 904-912Crossref PubMed Google Scholar). Microsensors were used in a self-referencing format to obtain a directional flux of oxygen by individual cells and monolayers, which is detected as a difference in current (Δfemptoamperes). The advantage of this method is that it minimizes noise and random drift in the measurements, factors that limit the use of standard oxygen electrodes (33Land S.C. Porterfield D.M. Sanger R.H. Smith P.J. J. Exp. Biol. 1999; 202: 211-218Crossref PubMed Google Scholar). Enzyme Assays—NAD(P)H:paraquat oxidoreductase activity was assayed in cell lysates by quantifying ROI generated via redox cycling of paraquat (36Bus J.S. Aust S.D. Gibson J.E. Biochem. Biophys. Res. Commun. 1974; 58: 749-755Crossref PubMed Scopus (475) Google Scholar, 37Winterbourn C.C. FEBS Lett. 1981; 128: 339-342Crossref PubMed Scopus (87) Google Scholar, 38Tampo Y. Tsukamoto M. Yonaha M. Free Radic Biol. Med. 1999; 27: 588-595Crossref PubMed Scopus (52) Google Scholar), by the paraquat-dependent consumption of oxygen or NADPH in the enzyme assay (39Zhang Z. Yu J. Stanton R.C. Anal. Biochem. 2000; 285: 163-167Crossref PubMed Scopus (98) Google Scholar), and by the NADPH-dependent formation of paraquat radical (36Bus J.S. Aust S.D. Gibson J.E. Biochem. Biophys. Res. Commun. 1974; 58: 749-755Crossref PubMed Scopus (475) Google Scholar, 37Winterbourn C.C. FEBS Lett. 1981; 128: 339-342Crossref PubMed Scopus (87) Google Scholar, 40Weidauer E. Morke W. Foth H. Bromme H.J. Arch. Toxicol. 2002; 76: 89-95Crossref PubMed Scopus (6) Google Scholar). To prepare cell lysates for the assays, cells were gently scraped from the culture dishes, resuspended in phosphate-buffered saline, and sonicated on ice with three 15-s bursts and 1 min of cooling on ice between each sonication burst. Disrupted cells were centrifuged (3000 × g, 10 min) to remove cellular debris, and the supernatants were either assayed immediately for enzyme activity or further used to purify and characterize the paraquat reductase (see further below). Unless otherwise specified, standard reaction mixes contained 50 mm phosphate buffer, pH 7.4, 0.5 mm NADPH or NADH, 10–100 μg of cell lysate protein, and 1–1000 μm paraquat in 0.1 ml. All enzyme assays were run at 37 °C. Hydrogen peroxide production was assayed in 100-μl reaction mixes supplemented with 25 μm 10-acetyl-3,7-dihydroxyphenoxazine and 0.1 units/sample of horseradish peroxidase as previously described (41Towne V. Will M. Oswald B. Zhao Q. Anal. Biochem. 2004; 334: 290-296Crossref PubMed Scopus (97) Google Scholar). The fluorescent product resorufin was quantified using an HTS 7000 Plus Bio Assay Reader (PerkinElmer Life Sciences) fitted with a 540-nm excitation filter and a 595-nm emission filter. Kinetic assays were performed in the presence of increasing concentrations of paraquat, and fluorescence of resorufin was measured every 2.5 min for 30 min. The rate of hydrogen peroxide formation was calculated based on a standard curve generated with hydrogen peroxide in a concentration range of 0.5–30 μm. Initial velocity studies of the NAD(P)H oxidase activity were performed as described previously (42Vetrano A.M. Heck D.E. Mariano T.M. Mishin V. Laskin D.L. Laskin J.D. J. Biol. Chem. 2005; 280: 35372-35381Abstract Full Text Full Text PDF PubMed Scopus (117) Google Scholar). In some experiments purified rat liver thioredoxin reductase (5 units/ml) or recombinant enzyme (9.8 units/ml) was added in place of cell lysates. Superoxide anion production was assayed by its ability to oxidize hydroethidine to 2-hydroxyethidium cation in the reaction mix; the product was detected using HPLC with fluorescence detection as described by Zhao et al. (43Zhao H. Joseph J. Fales H.M. Sokoloski E.A. Levine R.L. Vasquez-Vivar J. Kalyanaraman B. Proc. Natl. Acad. Sci. U. S. A. 2005; 102: 5727-5732Crossref PubMed Scopus (487) Google Scholar) with minor modifications in the chromatography protocol. HPLC was carried out using a Luna C18 (2Day B.J. Patel M. Calavetta L. Chang L.Y. Stamler J.S. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 12760-12765Crossref PubMed Scopus (168) Google Scholar), 3 μm, 250 × 2.0-mm column (Phenomenex, Torrance, CA) and a C18 guard column using a 0.2 ml/min flow rate. The column was equilibrated with 10% CH3CN in 0.1% trifluoroacetic acid. The formed products were separated by a linear increase in CH3CN concentration from 10 to 40% starting from 10 to 45 min. The signals from the detector were recorded and analyzed by CLASS-VP software (Shimadzu Scientific Instruments, Columbia, MD). Hydroxyl radicals were measured by the formation of 2-hydroxyterephthalate from terephthalate using HPLC with fluorescence detection (excitation 315 nm, emission 425 nm) as described by Mishin and Thomas (44Mishin V.M. Thomas P.E. Biochem. Pharmacol. 2004; 68: 747-752Crossref PubMed Scopus (26) Google Scholar). The generation of paraquat radicals and depletion of NADPH in reaction mixtures were quantified in 0.5-ml cuvettes using a Lambda 20 UV/visible spectrophotometer (PerkinElmer Life Sciences) scanning at 1 nm/s, recording at 1-nm intervals, and repeating the scan at 2.5-min intervals for 30 min. Paraquat radicals were quantified by increases in absorbance at 603 nm (ɛ603 = 1.20 × 104) (45Autor A.P. Biochemical Mechanisms of Paraquat Toxicity. 1977; (Ist Academic Press, New York): 21-38Google Scholar). Because paraquat radicals are sensitive to oxygen (46Farrington J.A. Ebert M. Land E.J. Fletcher K. Biochim. Biophys. Acta. 1973; 314: 372-381Crossref PubMed Scopus (487) Google Scholar), experiments were run in filled cuvettes covered tightly with Parafilm. NADPH depletion was measured by decreases in absorbance at 340 nm (39Zhang Z. Yu J. Stanton R.C. Anal. Biochem. 2000; 285: 163-167Crossref PubMed Scopus (98) Google Scholar). A polarographic system fitted with a Clark oxygen electrode (Yellow Springs Instruments, Yellow Springs, OH) was used to characterize oxygen utilization during enzymatic redox cycling of paraquat. Purification Studies—Lysates containing enzyme activity were centrifuged in an Eppendorf 5417R high speed centrifuge at 12,000 × g for 10 min to sediment membrane and mitochondrial fractions. The resulting supernatant was centrifuged in a Beckman L7–55 ultracentrifuge at 100,000 × g for 1 h, and the microsomal and post-microsomal supernatant fractions were collected. Approximately 40% of the paraquat oxidoreductase activity was contained in the post-microsomal supernatant fractions, whereas the remaining activity was present in membrane and microsomal fractions. The post-mitochondrial supernatant fractions were further purified by NADPH affinity chromatography as described by Wolff et al. (47Wolff D.J. Lubeskie A. Li C. Arch. Biochem. Biophys. 1997; 338: 73-82Crossref PubMed Scopus (20) Google Scholar). Briefly, 2′,5′-ADP-agarose was added (0.2 volume of settled resin per volume of post-microsomal supernatant), and the suspension was rocked in a cold room for 1 h and then centrifuged at 2000 × g for 5 min. The supernatant was discarded, and the resin was washed five times with phosphate-buffered saline containing 0.1% Nonidet P-40. The resin was then washed with phosphate-buffered saline containing 0.1% Nonidet P-40 and 5 mm NADPH. The NADPH eluate was concentrated using an Amicon Ultra centrifugal filter (Millipore, Billerica, MA) and then fractionated by size exclusion chromatography on a Superose 12 HR 10/30 column (GE Healthcare) in phosphate-buffered saline containing 0.1% Nonidet P-40, pH 7.4, at a flow rate of 0.3 ml/min. Absorbance of effluent fractions was monitored at 280 nm. The column was previously calibrated with Bio-Rad gel filtration standard proteins (thyroglobulin, Mr 60,000; bovine globulin, Mr 158,000; chicken ovalbumin, Mr 44,000). Fractions containing enzyme activity were analyzed on 10% SDS-polyacrylamide gels. Sequence analysis of protein bands in the gels were performed as previously described (48Senn H. Eugster A. Otting G. Suter F. Wuthrich K. Eur. Biophys. J. 1987; 14: 301-306Crossref PubMed Scopus (35) Google Scholar) and analyzed using NCBI Blast algorithms. Thioredoxin Reductase Assay—Thioredoxin reductase activity of the purified paraquat reductase or the recombinant enzyme was assayed by the reduction of 5,5′-dithiobis(2-nitrobenzoic acid) (DTNB) in the presence of NADPH as described by Kang et al. (49Kang H.J. Hong S.M. Kim B.C. Park E.H. Ahn K. Lim C.J. Mol. Cell. 2006; 22: 113-118Google Scholar). The assay mixture contained 0.2 m phosphate buffer, pH 7.4, 1 mm EDTA, 0.25 mm NADPH, 1 mm DTNB, and appropriate concentrations of purified recombinant thioredoxin reductase or lung cell extract. Enzyme activity was followed by increases in absorbance at 412 nm. In some experiments, 100 μm 1-chloro-2,4-dinitrobenzene or sodium aurothiomalate (ATM), inhibitors of mammalian thioredoxin reductase, were added the reaction mix. Purification of Thioredoxin Reductase—A construct containing human cytosolic thioredoxin reductase (hTR1) in which the TGA (Sec) codon was replaced with the TGC codon (Sec498Cys) cloned into the PET 28a+ vector (Novagen) was kindly provided by Anton Turanov (University of Nebraska). The thioredoxin reductase was purified from E. coli (BL21(DE3), Novagen) transformed with the construct and induced to express the enzyme as previously described (50Turanov A.A. Su D. Gladyshev V.N. J. Biol. Chem. 2006; 281: 22953-22963Abstract Full Text Full Text PDF PubMed Scopus (71) Google Scholar). Purity of the enzyme was confirmed by fractionation of the purified protein on a 10% SDS-polyacrylamide gel followed by Coomassie staining. Enzymatic activity was confirmed by measuring the enzymatic reduction of DTNB and inhibition of enzyme activity with 1-chloro-2,4-dinitrobenzene or ATM (51Nordberg J. Arner E.S. Free Radic Biol. Med. 2001; 31: 1287-1312Crossref PubMed Scopus (2220) Google Scholar, 52Gromer S. Arscott L.D. Williams Jr., C.H. Schirmer R.H. Becker K. J. Biol. Chem. 1998; 273: 20096-20101Abstract Full Text Full Text PDF PubMed Scopus (448) Google Scholar). Characterization of Lung Epithelial Cell Oxygen Flux—In initial experiments we used the polarographic self-referencing microsensor to characterize oxygen utilization by lung epithelial cells. When the sensor was placed 1–5 μm from the cells, an oxygen gradient could readily be detected (Figs. 1, A and B). By moving the probe away from the cells, the gradient was mapped. Oxygen flux was detectable at distances up to 50 μm from individual cells and 400–500 μm from confluent monolayers (Fig. 1B and not shown). The fact that oxygen gradients could be detected in areas surrounding the cells indicates that oxygen consumption by the cells is greater than the amount of oxygen available by its diffusion through the medium to the cells. Treatment of the cells with mitochondrial electron transport chain inhibitors rotenone (complex I), antimycin A (complex III), or potassium cyanide (complex IV) markedly reduced the oxygen gradients surrounding the cells, indicating that the oxygen consumption was mainly mediated by mitochondrial respiration (Figs. 1, panel C, and 2 and not shown). This conclusion is supported by our findings that the mitochondrial protonophore uncouplers, carbonyl cyanide p-trifluoromethoxyphenylhydrazone and carbonyl cyanide m-chlorophenylhydrazone, enhanced oxygen uptake by the cells (Figs. 1D and 2B). In further experiments we examined the effects of paraquat on oxygen flux into lung cells. The addition of 100 μm paraquat to the cells resulted in a marked increase (2–4-fold) in oxygen flux that was evident within 15–30 s and persisted for at least 30 min (Fig. 2A); smaller increases in oxygen flux (∼1.5-fold) were observed with 10 μm paraquat (not shown). Neither antimycin A nor potassium cyanide, which block mitochondrial respiration, altered paraquat-induced oxygen flux (Figs. 2, panels A and D, and 3). These data indicate that the effects of paraquat are independent of mitochondrial respiration. Pretreatment of lung cells with inhibitors of mitochondrial respiration also had no effect on paraquat-induced oxygen flux (Fig. 2C and not shown). In contrast, carbonyl cyanide p-trifluoromethoxyphenylhydrazone and carbonyl cyanide m-chlorophenylhydrazone-induced increases in oxygen flux were completely blocked by potassium cyanide, demonstrating that the actions of the mitochondrial protonophores and paraquat are distinct (Fig. 2B and not shown). Paraquat has also been reported to stimulate the diaphorase activity of nitric-oxide synthase, a process inhibited by the nitric-oxide synthase inhibitor nitroarginine methyl ester (NAME) (2Day B.J. Patel M. Calavetta L. Chang L.Y. Stamler J.S. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 12760-12765Crossref PubMed Scopus (168) Google Scholar). We found that treatment of the lung epithelial cells with NAME had no effect on mitochondrial or paraquat-induced oxygen flux (Fig. 2D and not shown). These data suggest that nitric-oxide synthase does not affect paraquat-induced oxygen uptake. It is well recognized that several NADPH oxidases, including cytochrome P450 reductase, contribute to extra-mitochondrial oxygen utilization (53Cadenas E. Davies K.J. Free Radic. Biol. Med. 2000; 29: 222-230Crossref PubMed Scopus (2409) Google Scholar, 54Mason H.S. Adv. Enzymol. Relat. Subj. Biochem. 1957; 19: 79-233PubMed Google Scholar). Paraquat is known to redox cycle via cytochrome P450 reductase; whether it is a substrate for NADPH oxidase is unknown (6Bonneh-Barkay D. Reaney S.H. Langston W.J. Di Monte D.A. Mol. Brain Res. 2005; 134: 52-56Crossref PubMed Scopus (135) Google Scholar). We found that diphenyleneiodonium (DPI), a non-selective inhibitor of FAD-dependent enzymes, including NADPH oxidase, readily blocked paraquat-induced oxygen flux into the cells without affecting mitochondrial respiration (Fig. 4A). This effect was independent of the order of addition of paraquat and DPI to the cultures (compare Figs. 4, A and B). Taken together, these data indicate that paraquat-induced alterations in oxygen uptake are dependent on an FAD-containing oxidoreductase activity in the cells. To eliminate the possibility that cytochrome P450 reductase was responsible for paraquat-induced increases in oxygen flux, we analyzed the effects of the herbicide in cells overexpressing the enzyme. For these studies we used CHO cells stably transfected with mouse CYP450 reductase that have 35-fold more enzyme activity when compared with wild type control CHO cells (31Han J.F. Wang S.L. He X.Y. Liu C.Y. Hong J.Y. Toxicol. Sci. 2006; 91: 42-48Crossref PubMed Scopus (41) Google Scholar). 3J. P. Gray, D. E. Heck, V. Mishin, P. J. S. Smith, J.-Y. Hong, M. Thiruchelvam, D. A. Cory-Slechta, D. L. Laskin, and Jeffrey D. Laskin, unpublished information. As observed with lung epithelial cells, paraquat caused a similar increase in oxygen flux into both wild type and cytochrome P450 reductase-overexpressing cells. Subsequent treatment of the cells with potassium cyanide reduced oxygen flux due to mitochondrial respiration (Fig. 5, panels A and B). The fact that the different cell types responded similarly to paraquat indicates that oxygen uptake by the cells induced by paraquat was independent of cytochrome P450 reductase activity. Effects of Paraquat on Lung Epithelial Cell NADPH Oxidase—NADPH oxidase catalyzes the transfer of electrons from NADPH to oxygen resulting in the formation of superoxide anion. Superoxide anion rapidly dismutates into hydrogen peroxide and, in the presence of redox active metals, is a source of cytotoxic hydroxyl radicals (for review, see Refs. 10McCord J.M. Am. J. Med. 2000; 108: 652-659Abstract Full Text Full Text PDF PubMed Scopus (1078) Google Scholar and 11Mason R.P. Environ. Health Perspect. 1990; 87: 237-243Crossref PubMed Scopus (61) Google Scholar). NADPH oxidase enzyme activity can readily be quantified by the NADPH-dependent consumption of oxygen, utilization of NADPH, and the production of ROI due to redox cycling of paraquat (36Bus J.S. Aust S.D. Gibson J.E. Biochem. Biophys. Res. Commun. 1974; 58: 749-755Crossref PubMed Scopus (475) Google Scholar, 37Winterbourn C.C. FEBS Lett. 1981; 128: 339-342Crossref PubMed Scopus (87) Google Scholar, 38Tampo Y. Tsukamoto M. Yonaha M. Free Radic Biol. Med. 1999; 27: 588-595Crossref PubMed Scopus (52) Google Scholar, 39Zhang Z. Yu J. Stanton R.C. Anal. Biochem. 2000; 285: 163-167Crossref PubMed Scopus (98) Google Scholar). Mitochondria-free lysates prepared from lung epithelial cells contained very low levels of NADPH oxidase enzyme activity, as measured by oxygen consumption using a Clark oxygen electrode (Fig. 5). The addition of paraquat to the reaction mix caused a marked increase in enzyme activity that was dependent on NADPH. Oxygen consumption by this enzyme was inhibited by DPI (Fig. 5). These findings are consistent with our microsensor studies in intact cells and support the concept that the actions of paraquat are mediated by an FAD-containing NADPH oxidase. In further studies we characterized the paraquat-stimulated NADPH oxidase activity in lung cell lysates. A one-electron reduction of paraquat by this enzyme would be expected to generate the paraquat radical (see Reaction 1). This radical is stable at low oxygen tension and can be quantified spectrophotometrically by its absorption maximum at 603 nm (45Autor A.P. Biochemical