Molecular characterization of NAD(P)H:quinone oxidoreductase of tobacco leaves

Summary The NAD(P)H:quinone oxidoreductase activity of tobacco leaves is catalyzed by a soluble flavoprotein [NAD(P)H-QR] and membrane-bound forms of the same enzyme. In particular, the activity associated with the plasma membrane cannot be released by hypoosmotic and salt washing of the vesicles, suggesting a specific binding. The products of the plasma-membrane-bound quinone reductase activity are fully reduced hydroquinones rather than semi-quinone radicals. This peculiar kinetic property is common with soluble NAD(P)H-QR, plasma-membrane-bound NAD(P)H:quinone reductase purified from onion roots, and animal DT-diaphorase. These and previous results demonstrate that soluble and plasma-membrane-bound NAD(P)H:quinone reductases are strictly related flavo-dehydrogenases which seem to replace DT-diaphorase in plant tissues. Following purification to homogeneity, the soluble NAD(P)H-QR from tobacco leaves was digested. Nine peptides were sequenced, accounting for about 50% of NAD(P)H-QR amino acid sequence. Although one peptide was found homologous to animal DT-diaphorase and another one to plant monodehydroascorbate reductase, native NAD(P)H-QR does not seem to be structurally similar to any known flavoprotein.Abbreviations MDAR monodehydroascorbate reductase - PM plasma membrane - NAD(P)H-QR NAD(P)H:quinone oxidoreductase - DPI diphenylene iodonium - DQ duroquinone - CoQ₂ coenzyme Q₂     

Purification and Characterization of an NAD(P)H:Quinone Oxidoreductase from Glycine Max Seedlings

Abstract

An NAD(P)H:(quinone acceptor) oxidoreductase (EC 1.6.99.2) was purified from Glycine max seedlings by means of chromatographic procedures. After 1371-fold purification, the enzyme showed a single band in IEF corresponding to an isoelectric point of 6.1. A single band was also found in native-PAGE both by activity staining and Coomassie brilliant blue staining. The molecular mass determined in SDS-PAGE was 21900 Da, while in HPLC gel-filtration it was 61000 Da. The NAD(P)H:quinone oxidoreductase was able to use NADH or NADPH as the electron donor. Among the artificial quinones which are reduced by this enzyme, 6-hydroxydopa- and 6-hydroxydopamine-quinone are of particular interest because of their neurotoxic effects.     

A two-domain structure for the two subunits of NAD(P)H:quinone acceptor oxidoreductase.

NAD(P)H:quinone acceptor oxidoreductase (EC 1.6.99.2) (DT-diaphorase) is a FAD-containing reductase that catalyzes a unique 2-electron reduction of quinones. It consists of 2 identical subunits. In this study, it was found that the carboxyl-terminal portion of the 2 subunits can be cleaved by various proteases, whereas the amino-terminal portion cannot. It was also found that proteolytic digestion of the enzyme can be blocked by the prosthetic group FAD, substrates NAD(P)H and menadione, and inhibitors dicoumarol and phenindione. Interestingly, chrysin and Cibacron blue, 2 additional inhibitors, cannot protect the enzyme from proteolytic digestion. The results obtained from this study indicate that the subunit of the quinone reductase has a 2-domain structure, i.e., an amino-terminal compact domain and a carboxyl-terminal flexible domain. A structural model of the quinone reductase is generated based on results obtained from amino-terminal and carboxyl-terminal protein sequence analyses and electrospray mass spectral analyses of hydrolytic products of the enzyme generated by trypsin, chymotrypsin, and Staphylococcus aureus protease. Furthermore, based on the data, it is suggested that the binding of substrates involves an interaction between 2 structural domains.     

Purification and crystallization of rat liver NAD(P)H:(quinone-acceptor) oxidoreductase by cibacron blue affinity chromatography: identification of a new and potent inhibitor

Cytosolic NAD(P)H:(quinone-acceptor) oxidoreductase (EC 1.6.99.2) is a widely distributed, FAD-containing enzyme that catalyzes the obligatory two-electron reduction of quinones. Cibacron Blue is an inhibitor of this enzyme comparable in potency to dicoumarol. Pure quinone reductase was obtained from the livers of Sudan II (1-[2,4-dimethylphenylazo]-2-naphthol)-treated rats in a single step by Cibacron Blue-agarose chromatography. Cibacron Blue is a competitive inhibitor with respect to NADH (Ki = 170 nM) and is a noncompetitive inhibitor with respect to menadione (Ki = 540 nM). Addition of Cibacron Blue to quinone reductase resulted in a decrease and red shift of the enzyme-bound FAD peak at 450 nm. The titration of the absorbance changes for both FAD and Cibacron Blue could be fitted to curves describing an equilibrium binding equation with a KD of 300 nM and one binding site per enzyme subunit. Furthermore, the Cibacron Blue difference spectrum that resulted from binding to quinone reductase was abolished by dicoumarol. Significant amino acid homology between quinone reductase and the nucleotide binding regions of enzymes that bind to Cibacron Blue was found. These data indicate that Cibacron Blue is a useful ligand for the purification of quinone reductase and a new probe for its NAD(P)H binding site. Conditions for crystallizing rat liver quinone reductase are also described.     

Properties of a coenzyme, pyrroloquinoline quinone: generation of an active oxygen species during a reduction-oxidation cycle in the presence of NAD(P)H and O2

The oxidation of NAD(P)H by pyrroloquinoline quinone (PQQ) was non-enzymatically carried out at physiological pH in the presence of O2. The PQQ-NAD(P)H system requires about 1 mol of O2 for the oxidation of 1 mol of NAD(P)H. The oxidation of NAD(P)H occurred at a pseudo-first-order rate with respect to NAD(P)H and was of zero order with respect to PQQ concentration in in the presence of O2: k0[PQQ] [NAD(P)H] = k1 [NAD(P)H], where k0[PQQ] = k1, in which [PQQ] represents the initial concentration of PQQ. k0 values for NADH and NADPH were 3.4.10(2) M-1.min-1 and 2.0.10(2) M-1.min-1, respectively, at 25 degrees C and at 258 microM O2 (initial concentration). The system produced O-2, probably by the interaction of PQQ.H and/or NAD(P).with O2, during the oxidation of NAD(P)H. PQQH2 and PQQ.H were easily oxidized to PQQ in the presence of O2, yielding H2O2.     

Preliminary crystallographic data for NAD(P)H quinone reductase isolated from the Walker 256 rat carcinoma cell line

An NAD(P)H quinone reductase isolated from Walker rat 256 carcinoma cells has been crystallized in a form suitable for high-resolution structural analysis. The crystals belong to orthorhombic space group P2(1)2(1)2(1) with cell parameters a = 168.15 A, b = 105.09 A and c = 67.38 A and contain four monomeric or two dimeric enzyme molecules per asymmetric unit. Diffraction extends beyond 2.3 A resolution.     

Nucleotide and deduced amino acid sequence of a human cDNA (NQO2) corresponding to a second member of the NAD(P)H:quinone oxidoreductase gene family. Extensive polymorphism at the NQO2 gene locus on chromosome 6.

NAD(P)H:quinone oxidoreductases (NQOs) are flavoproteins that catalyze the oxidation of NADH or NADPH by various quinones and oxidation-reduction dyes. We have previously described a complementary DNA that encodes a dioxin-inducible cytosolic form of human NAD(P)H:quinone oxidoreductase (NQO1). In the present report we describe the nucleotide sequence and deduced amino acid sequence for a cDNA clone that is likely to encode a second form of NAD(P)H:quinone oxidoreductase (NQO2) which was isolated by screening a human liver cDNA library by hybridization with a NQO1 cDNA probe. The NQO2 cDNA is 976 nucleotides long and encodes a protein of 231 amino acids (Mr = 25,956). The human NQO2 cDNA and protein are 54% and 49% similar to human liver cytosolic NQO1 cDNA and protein, respectively. COS1 cells transfected with NQO2 cDNA showed a 5-7-fold increase in NAD(P)H:quinone oxidoreductase activity as compared to nontransfected cells when either 2,6-dichlorophenolindophenol or menadione was used as substrate. Western blot analysis of the expressed NQO1 and NQO2 cDNA proteins showed cross-reactivity with rat NQO1 antiserum, indicating that NQO1 and NQO2 proteins are immunologically related. Northern blot analysis shows the presence of one NQO2 mRNA of 1.2 kb in control and 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) treated human hepatoblastoma Hep-G2 cells and that TCDD treatment does not lead to enhanced levels of NQO2 mRNA as it does for NQO1 mRNA. Southern blot analysis of human genomic DNA suggests the presence of a single gene approximately 14-17 kb in length. The NQO2 gene locus is highly polymorphic as indicated by several restriction fragment length polymorphisms detected with five different restriction enzymes.(ABSTRACT TRUNCATED AT 250 WORDS)     

Photodependent inhibition of rat liver NAD(P)H:quinone acceptor oxidoreductase by (A)-2-azido-NAD+ and (A)-8-azido-NAD

Two photoaffinity analogues of NAD+, (A)-2-azido-NAD+ [nicotinamide 2-azidoadenine dinucleotide] and (A)-8-azido-NAD+ [nicotinamide 8-azidoadenine dinucleotide], have been synthesized, and their reactivities with the rat liver NAD(P)H:quinone acceptor oxidoreductase have been investigated. The reduce nicotinamide nucleotide probes, (A)-2-azido-NADH and (A)-8-azido-NADH, were shown to be substrates of the quinone reductase. This enzyme was inhibited by (A)-8-azido-NADH, were shown to be substrates of the quinone reductase. This enzyme was inhibited by (A)-2-azido-NAD+ and (A)-8-azido-NAD+ in a photodependent manner, and the inhibition of the enzyme could be prevented by the presence of nicotinamide nucleotide substrates during photolysis. (A)-2-Azido-NAD+ was demonstrated to be a more potent inhibitor than (A)-8-azido-NAD+. In addition, the photodependent inhibition by (A)-8-azido-NAD+ increased when menadione, the substrate of the enzyme, was present during the photolysis, while menadione protected the enzyme from the photodependent inhibition by (A)-2-azido-NAD+. These results indicate that these two NAD+ analogues can be used to identify the nicotinamide nucleotide binding site of this quinone reductase and that they probably bind to the enzyme in different fashions.     

NAD(P)H fluorescence imaging of mitochondrial metabolism in contracting Xenopus skeletal muscle fibers: effect of oxygen availability.

The blue autofluorescence (351 nm excitation, 450 nm emission) of single skeletal muscle fibers from Xenopus was characterized to be originating from mitochondrial NAD(P)H on the basis of morphological and functional correlations. This fluorescence signal was used to estimate the oxygen availability to isolated single Xenopus muscle fibers during work level transitions by confocal microscopy. Fibers were stimulated to generate two contractile periods that were only different in the PO2 of the solution perfusing the single fibers (PO2 of 30 or 0-2 Torr; pH = 7.2). During contractions, mean cellular NAD(P)H increased significantly from rest in the low PO2 condition with the core (inner 10%) increasing to a greater extent than the periphery (outer 10%). After the cessation of work, NAD(P)H decreased in a manner consistent with oxygen tensions sufficient to oxidize the surplus NAD(P)H. In contrast, NAD(P)H decreased significantly with work in 30 Torr PO2. However, the rate of NAD(P)H oxidation was slower and significantly increased with the cessation of work in the core of the fiber compared with the peripheral region, consistent with a remaining limitation in oxygen availability. These results suggest that the blue autofluorescence signal in Xenopus skeletal muscle fibers is from mitochondrial NAD(P)H and that the rate of NAD(P)H oxidation within the cell is influenced by extracellular PO2 even at high extracellular PO2 during the contraction cycle. These results also demonstrate that although oxygen availability influences the rate of NAD(P)H oxidation, it does not prevent NAD(P)H from being oxidized through the process of oxidative phosphorylation at the onset of contractions.     

Induction of epidermal NAD(P)H:quinone reductase by chemical carcinogens: a possible mechanism for the detoxification

NAD(P)H:quinone reductase, which plays an important role in the detoxification of carcinogenic metabolites as well as oxidative cellular damage, was found to be present in epidermal cytosol where its specific activity far exceeds (140-160%) the corresponding hepatic value. The effect of topical application of crude coal tar, 3-methylcholanthrene and polychlorinated biphenyl Aroclor 1254, on epidermal and hepatic cytosolic NAD(P)H:quinone reductase activities was investigated in neonatal rats, Sencar and athymic nude mice. A single topical application of each agent resulted in significant increases in epidermal (185%-389%) and hepatic (150-255%) enzyme activities. This inducible enzyme may play an important role in the detoxification of reactive quinone species during the course of malignant neoplasia and against oxidative cellular damage in skin.     

Histochemical Detection of Quinone Reductase Activity In Situ Using LY 83583 Reduction and Oxidation

Abstract: The application of enzymatic staining techniques, using tetrazolium dyes, to aldehyde-treated brain sections has revealed the presence of NADPH-diaphorase activity attributed to nitric oxide synthase. When evaluating the specificity of the putative guanylyl cyclase inhibitor LY 83583, a robust and novel staining pattern was noted in epithelial, endothelial, and astrocytic cells when LY 83583 was included in the NADPH-diaphorase histochemical reaction. This LY 83583-dependent staining could be blocked by the NAD(P)H:quinone oxidoreductase inhibitor dicumarol. Based on its quinone structure, we hypothesized that LY 83583 was a substrate for the enzyme NAD(P)H:quinone oxidoreductase. Transfection of human embryonic kidney 293 cells with the rat liver isoform of NAD(P)H:quinone oxidoreductase resulted in robust NADPH- and LY 83583-dependent staining that was completely blocked by dicumarol and was not observed in untransfected cells. Analysis of transfected cell extracts and brain homogenates indicated that LY 83583 was a substrate for NAD(P)H:quinone oxidoreductase, with a K _m similar to the well-characterized substrate menadione. Sensitivity of the nitroblue tetrazolium reduction to superoxide dismutase indicated that the reduction of LY 83583 by NAD(P)H:quinone oxidoreductase leads to superoxide generation. The localization of NAD(P)H:quinone oxidoreductase activity to astrocytic cells suggests a role for glia in combating oxidative insults to brain and in activating quinone-like drugs such as LY 83583.     

Regeneration of lipophilic antioxidants by NAD(P)H:quinone oxidoreductase 1

Summary.  The aim of this work was to study the activity of NAD(P)H:(quinone acceptor) oxidoreductase 1 (EC 1.6.99.2) in the regeneration of lipophilic antioxidants, alpha-tocopherol, and reduced-coenzyme Q analogs. First, we tested whether or not two isoforms of the NAD(P)H:(quinone acceptor) oxidoreductase 1 designated as “hydrophilic” and “hydrophobic” (H. J. Prochaska and P. Talalay, Journal of Biological Chemistry 261: 1372–1378, 1986) show differential enzyme activities towards hydrophilic or hydrophobic ubiquinone homologs. By chromatography on phenyl Sepharose, we purified the two isoforms from pig liver cytosol and measured their reduction of several ubiquinone homologs of different side chain length. We also studied by electron paramagnetic resonance the effect of NAD(P)H:(quinone acceptor) oxidoreductase 1 on steady-state levels of chromanoxyl radicals generated by linoleic acid and lipooxygenase and confirmed the enzyme's ability to protect alpha-tocopherol against oxidation induced with H₂O₂-Fe²⁺. Our results demonstrated that the different hydrophobicities of the isoforms do not reflect different reactivities towards ubiquinones of different side chain length. In addition, electron paramagnetic resonance studies showed that in systems containing the reductase plus NADH, levels of chromanoxyl radicals were dramatically reduced. Morever, in the presence of oxidants, alpha-tocopherol was preserved by NAD(P)H:(quinone acceptor) oxidoreductase 1, supporting our hypothesis that regeneration of alpha-tocopherol may be one of the physiologic functions of this enzyme. Received May 20, 2002; accepted September 20, 2002; published online May 21, 2003 RID="*" ID="*" Correspondence and reprints: Departamento de Biología Celular, Fisiología e Inmunología, Facultad de Ciencias, Edificio Severo Ochoa, Campus de Rabanales, Universidad de Córdoba, 14014 Córdoba, Spain.     

Dissecting the Diphenylene Iodonium-Sensitive NAD(P)H:Quinone Oxidoreductase of Zucchini Plasma Membrane

Quinone oxidoreductase activities dependent on pyridine nucleotides are associated with the plasma membrane (PM) in zucchini (Cucurbita pepo L.) hypocotyls. In the presence of NADPH, lipophilic ubiquinone homologs with up to three isoprenoid units were reduced by intact PM vesicles with a Km of 2 to 7 [mu]M. Affinities for both NADPH and NADH were similar (Km of 62 and 51 [mu]M, respectively). Two NAD(P)H:quinone oxidoreductase forms were identified. The first, labeled as peak I in gel-filtration experiments, behaves as an intrinsic membrane complex of about 300 kD, it slightly prefers NADH over NADPH, it is markedly sensitive to the inhibitor diphenylene iodonium, and it is active with lipophilic quinones. The second form (peak II) is an NADPH-preferring oxidoreductase of about 90 kD, weakly bound to the PM. Peak II is diphenylene iodonium-insensitive and resembles, in many properties, the soluble NAD(P)H:quinone oxidoreductase that is also present in the same tissue. Following purification of peak I, however, the latter gave rise to a quinone oxidoreductase of the soluble type (peak II), based on substrate and inhibitor specificities and chromatographic and electrophoretic evidence. It is proposed that a redox protein of the same class as the soluble NAD(P)H:quinone oxidoreductase (F. Sparla, G. Tedeschi, and P. Trost [1996] Plant Physiol. 112:249-258) is a component of the diphenylene iodonium-sensitive PM complex capable of reducing lipophilic quinones.     

Antioxidant response induced by serum withdrawal protects HL-60 cells against inhibition of NAD(P)H:quinone oxidoreductase 1

We have previously shown that inhibition of NAD(P)H:quinone acceptor oxidoreductase 1 with dicoumarol decreases growth and viability of HL-60 cells in the absence of serum. Here we demonstrate that culturing HL-60 cells in serum-free medium in the presence of dicoumarol results in a significant potentiation of apoptosis. However, when cells were preincubated for 24 h without serum before they were treated with dicoumarol, the effect of the inhibitor on cell growth and death was much lower. We have investigated cellular changes induced in HL-60 cells by removal of serum that could account for protection against the effects of dicoumarol. Serum removal induced significant increases of NAD(P)H:quinone acceptor oxidoreductase 1, particularly at 32 h after serum withdrawal. Total amounts of ubiquinone in cells were unchanged but, its reduction state paralleled the observed increase in quinone reductase activity. Levels of the antiapoptotic protein Bcl-2 were also significantly increased after serum removal. Our results indicate that removal of serum evokes an antioxidant protective response that make HL-60 cells less sensitive to cell death induced by inhibition of NAD(P)H:quinone acceptor oxidoreductase 1 with dicoumarol.     

NRH:quinone oxidoreductase2 (NQO2)

The quinone oxidoreductases [NAD(P)H:quinone oxidoreductase1 (NQO1) and NRH:quinone oxidoreductase2 (NQO2)] are flavoproteins. NQO1 is known to catalyse metabolic detoxification of quinones and protect cells from redox cycling, oxidative stress and neoplasia. NQO2 is a 231 amino acid protein (25956 mw) that is 43 amino acids shorter than NQO1 at its carboxy-terminus. The human NQO2 cDNA and protein are 54 and 49% similar to the human liver cytosolic NQO1 cDNA and protein. Recent studies have revealed that NQO2 differs from NQO1 in its cofactor requirement. NQO2 uses dihydronicotinamide riboside (NRH) rather than NAD(P)H as an electron donor. Another difference between NQO1 and NQO2 is that NQO2 is resistant to typical inhibitors of NQO1, such as dicoumarol, Cibacron blue and phenindone. Flavones, including quercetin and benzo(a)pyrene, are known inhibitors of NQO2. Even though overlapping substrate specificities have been observed for NQO1 and NQO2, significant differences exist in relative affinities for the various substrates. Analysis of the crystal structure of NQO2 revealed that NQO2 contains a specific metal binding site, which is not present in NQO1. The human NQO2 gene has been precisely localized to chromosome 6p25. The human NQO2 gene locus is highly polymorphic. The NQO2 gene is ubiquitously expressed and induced in response to TCDD. Nucleotide sequence analysis of the NQO2 gene promoter revealed the presence of several cis-elements, including SP1 binding sites, CCAAT box, xenobiotic response element (XRE) and an antioxidant response element (ARE). The complement of these elements regulates tissue specific expression and induction of the NQO2 gene in response to xenobiotics and antioxidants. The in vivo role of NQO2 and its role in quinone detoxification remains unknown.     

Purification by cibacron blue F3GA dye affinity chromatography and comparison of NAD(P)H:quinone reductase (E.C.1.6.99.2) from rat liver cytosol and microsomes

The dicoumarol-sensitive NAD(P)H:quinone reductase (E.C.1.6.99.2), often referred to as DT-diaphorase, has been purified from both the cytosolic and microsomal fractions from rat liver using a novel, highly efficient, two-step purification procedure utilizing immobilized Cibacron Blue F3GA dye affinity chromatography as the principal step. Under the conditions reported here, this dye affinity resin, generally recognized as preferentially binding nucleotide-dependent proteins, was highly selective in the recovery of up to 95% of the NAD(P)H:quinone reductase directly from the cytosol as a preparation which was often greater than 90% pure. Further purification by gel exclusion chromatography resulted in pure protein preparations with final recoveries approaching 80%. Similar results were obtained during the purification of this quinone reductase activity from microsomal extracts. Evidence is presented which suggests that the enzyme isolated from each cellular fraction are highly homologous, if not identical; data are consistent with genetic evidence.     

In vivo role of NAD(P)H:quinone oxidoreductase 1 (NQO1) in the regulation of intracellular redox state and accumulation of abdominal adipose tissue

NAD(P)H:quinone oxidoreductase 1 (NQO1) is a flavoprotein that utilizes NAD(P)H as an electron donor, catalyzing the two-electron reduction and detoxification of quinones and their derivatives. NQO1-/- mice deficient in NQO1 activity and protein were generated in our laboratory (Rajendirane, V., Joseph, P., Lee, Y. H., Kimura, S., Klein-Szanto, A. J. P., Gonzalez, F. J., and Jaiswal, A. K. (1998) J. Biol. Chem. 273, 7382-7389). Mice lacking a functional NQO1 gene (NQO1-/-) were born normal and reproduced adeptly as the wild-type NQO1+/+ mice. In the present report, we show that NQO1-/- mice exhibit significantly lower levels of abdominal adipose tissue as compared with the wild-type mice. The NQO1-/- mice showed lower blood levels of glucose, no change in insulin, and higher levels of triglycerides, beta-hydroxy butyrate, pyruvate, lactate, and glucagon as compared with wild-type mice. Insulin tolerance test demonstrated that the NQO1-/- mice are insulin resistant. The NQO1-/- mice livers also showed significantly higher levels of triglycerides, lactate, pyruvate, and glucose. The liver glycogen reserve was found decreased in NQO1-/- mice as compared with wild-type mice. The livers and kidneys from NQO1-/- mice also showed significantly lower levels of pyridine nucleotides but an increase in the reduced/oxidized NAD(P)H:NAD(P) ratio. These results suggested that loss of NQO1 activity alters the intracellular redox status by increasing the concentration of NAD(P)H. This leads to a reduction in pyridine nucleotide synthesis and reduced glucose and fatty acid metabolism. The alterations in metabolism due to redox changes result in a significant reduction in the amount of abdominal adipose tissue.     

Prevention of salt-induced renal injury by activation of NAD(P)H:quinone oxidoreductase 1, associated with NADPH oxidase

NADPH oxidase (NOX) is a predominant source of reactive oxygen species (ROS), and the activity of NOX, which uses NADPH as a common rate-limiting substrate, is upregulated by prolonged dietary salt intake. β-Lapachone (βL), a well-known substrate of NAD(P)H:quinone oxidoreductase 1 (NQO1), decreases the cellular NAD(P)H/NAD(P)(+) ratio via activation of NQO1. In this study, we evaluated whether NQO1 activation by βL modulates salt-induced renal injury associated with NOX-derived ROS regulation in an animal model. Dahl salt-sensitive (DS) rats fed a high-salt (HS) diet were used to investigate the renoprotective effect of NQO1 activation. βL treatment significantly lowered the cellular NAD(P)H:NAD(P)(+) ratio and dramatically reduced NOX activity in the kidneys of HS diet-fed DS rats. In accordance with this, total ROS production and expression of oxidative adducts also decreased in the βL-treated group. Furthermore, HS diet-induced proteinuria and glomerular damage were markedly suppressed, and inflammation, fibrosis, and apoptotic cell death were significantly diminished by βL treatment. This study is the first to demonstrate that activation of NQO1 has a renoprotective effect that is mediated by NOX activity via modulation of the cellular NAD(P)H:NAD(P)(+) ratio. These results provide strong evidence that NQO1 might be a new therapeutic target for the prevention of salt-induced renal injury.