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Department of Pharmaceutical Sciences and Cancer Center, School of Pharmacy, University of Colorado Health Sciences Center, Denver, Colorado (D.S., D.L.G., D.L.D., D.R.); and Department of Chemistry and Biochemistry, University of Denver, Denver, Colorado (J.Y.H., P.B., L.J.B.)
Received December 11, 2003; accepted February 5, 2004
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Abstract |
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), whereas the addition of superoxide dismutase greatly inhibited the rate of auto-oxidation. The ability of reduced NQO1 to react with suggested a role for NQO1 in scavenging , and this hypothesis was tested using established methods for production and detection. The addition of NQO1 in combination with NAD(P)H resulted in inhibition of dihydroethidium oxidation, pyrogallol auto-oxidation, and elimination of a potassium superoxide-generated ethoxycarbonyl-2-methyl-3,4-dihydro-2H-pyrrole-1-oxide: adduct signal (electron spin resonance). Kinetic parameters for the reduction of by NQO1 were estimated using xanthine/xanthine oxidase as the source of and after NQO1-dependent NADH oxidation at 340 nm. The ability of NQO1 to scavenge was also examined using cell sonicates prepared from isogenic cell lines containing no NQO1 activity (NQO1-) or very high levels of NQO1 activity (NQO1+). We demonstrated that addition of NAD(P)H and cell sonicate from NQO1+ but not NQO1- cells resulted in an increased level of scavenging could be inhibited by 5-methoxy-1,2-dimethyl-3-[(4-nitrophenoxy)methyl]indole-4,7-dione (ES936), a mechanism-based inhibitor of NQO1. NQO1 can generate hydroquinones that are redox active, and the scavenging activity of NQO1 may allow protection against at the site of hydroquinone generation. In addition, the scavenging activity of NQO1 may provide an additional level of protection against induced toxicity.
). NQO1 is a FAD-containing protein that exists as a homodimer and is biochemically characterized by its unique ability to use either NADH or NADPH as reducing cofactors and by its inhibition by the anticoagulant dicumarol (Ernster, 1967; Edwards et al., 1980). The enzyme is generally considered as a detoxification enzyme because of its ability to reduce reactive quinones and quinone-imines to less reactive and less toxic hydroquinones. The two-electron reduction of quinones also bypasses semiquinone production and thus prevents the generation of reactive oxygen species such as and by disproportionation hydrogen peroxide (Thor et al., 1982; Lind et al., 1990). The ability of NQO1 to deactivate many reactive species, including quinones, quinone-imines, and azo compounds, demonstrates its importance as a chemoprotective enzyme. The other major protective effect of NQO1 is to function as a cancer preventive enzyme, which has been recognized for more than 30 years (Huggins and Fukunishi, 1964). In humans, high levels of NQO1 have been found in most tissues except for liver (Siegel and Ross, 2000; Strassburg et al., 2002). Immunohistochemical studies have revealed that NQO1 is expressed primarily in epithelium, endothelium, and adipocytes (Siegel and Ross, 2000). In addition, NQO1 is also expressed at high levels in many epithelial-derived human tumors (Siegel et al., 1998; Siegel and Ross, 2000; Wilson et al., 2001).
The function(s) of NQO1 may not be restricted to metabolism of xenobiotic quinones. NQO1 may play an antioxidant role via the reduction of endogenous quinones such as vitamin E quinone (Siegel et al., 1997) and coenzyme Q10 (Beyer et al., 1996). Reduction of these compounds by NQO1 generates stable hydroquinones with excellent antioxidant properties. Other findings suggest that NQO1 may have an even broader antioxidant role. An important observation is that x-radiation and UV radiation, which are known to generate free radicals, are some of the most potent inducers of NQO1 expression in human cells (Boothman et al., 1993; Agrawal et al., 2001). The absence of NQO1 in human liver and moderately high expression throughout most other tissues suggest that the function for NQO1 may have evolved from primarily quinone detoxification to a more broadened antioxidant role. It has been proposed that NQO1 stabilizes p53 (Asher et al., 2001), and in recent studies, we have shown that NQO1 physically associates with the tumor suppressor protein p53 in human tumor cells and primary cultures (Anwar et al., 2003). p53 is a powerful transcription factor that up-regulates a number of proteins involved in protection against oxidant stress including proteins with homology to quinone oxidoreductases/NQO1 (Polyak et al., 1997). All of these findings suggest a more direct role for NQO1 in protection against oxidative stress than considered previously. In this work, we demonstrate that NQO1 has the ability to directly scavenge .
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Materials and Methods |
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). Recombinant human NQO1 was purified from Escherichia coli by Cibacron blue affinity chromatography as described previously (Beall et al., 1994). Purity was greater than 98% as determined by colloidal Brilliant Blue G staining (Sigma-Aldrich). Purified NQO1 had an activity of between 350 and 500 µmol of DCPIP/min/mg of protein and was stored in 25 mM Tris-HCL, pH 7.4, containing 250 mM sucrose. NQO1 activity was measured using dicumarol-inhibition of DCPIP reduction as described previously (Benson et al., 1980). In brief, NQO1 activity assays were performed on purified NQO1 and cell cytosols using the following conditions: 200 µM NADH and 40 µM DCPIP in 25 mM Tris-HCL containing 0.7 mg/ml bovine serum albumin (1-ml reaction volume). After the addition of purified protein or cytosol, the decrease in absorbance at 600 nm was monitored over 1 min at 27°C. Reactions were performed in the presence and absence of 10 µM dicumarol. Protein concentrations were determined using the method of Lowry et al. (1951).
Cell Lines. Construction of the human NQO1 transfected (stable) MDA468-NQ16 cell line from the parental MDA468 cell line has been described previously (Dehn et al., 2003). The NQO1 activity of the parental cell line was measured at less than 5 nmol of DCPIP/min/mg of protein, whereas the NQO1 activity of the MDA468/NQ16 cell line was greater than 2500 nmol of DCPIP/min/mg of protein. Both cell lines were grown in minimal essential medium supplemented with 10% (v/v) fetal bovine serum, 2 mM L-glutamine, 100 U/ml penicillin, and 100 µg/ml streptomycin at 37°C in a humidified atmosphere with 5% (v/v) CO2. Construction of the human NQO1 transfected (stable) CHO-DTD812 cell line from the parental Chinese hamster ovary (CHO) cell line has been described previously (Gustafson et al., 1996). No NQO1 activity or immunoreactive NQO1 protein could be detected in the parental CHO cell line, whereas the NQO1 activity of the CHO/812 cell line was greater than 3500 nmol DCPIP/min/mg of protein. Both CHO cell lines were grown in Ham's F-12 medium supplemented with 7% (v/v) fetal bovine serum, 20 mm HEPES, pH 7.4, 1 mM L-glutamine, 50 U/ml penicillin, and 50 µg/ml streptomycin at 37°C in a humidified atmosphere with 5% (v/v) CO2. For cytosol preparation, all cell lines were grown to three-quarters confluence in 100-mm plates, medium was removed, and the cells were washed with phosphate-buffered saline, pH 7.4. After rinsing, cells were scraped into phosphate-buffered saline and pelleted by centrifugation. The cell pellet was resuspended in 2.5 ml of Tris-HCL, pH 7.4, containing 250 mM sucrose, sonicated on ice for 5 s, and followed by centrifugation at 30,000g (15 min) to remove insoluble matter. Protein concentrations were determined on supernatants using the method of Lowry et al. (1951). For toxicity studies CHO cells were treated with freshly prepared pyrogallol dissolved in 20 mM HEPES, pH 8.0, for 2 h, after which the HEPES solution was replaced with fresh growth media, and the cells were allowed to divide for 4 days. The MTT-based cytotoxicity assay was performed as described previously (Dehn et al., 2003).
Auto-Oxidation of NQO1. The auto-oxidation of NQO1 was monitored in a stirred cell at 35°C using a Clark electrode (YSI Inc., Yellow Springs, OH). Reactions (4 ml) contained 50 mM potassium phosphate buffer, pH 7.4, 0.1 mM deferoxamine, and 200 µM NADH. Before the addition of NQO1 (400 µg), a 1-ml aliquot of reaction was removed for spectrophotometric analysis at 340 nm to determine the initial NADH concentration. Oxygen consumption was monitored over 15 min after which another 1-ml aliquot was immediately removed for NADH analysis. Reactions were run in the presence and absence of SOD (5 U/ml). Oxygen consumption was linear over the length of the experiment except for an initial lag period of approximately 1 min. To determine products (superoxide and hydrogen peroxide) generated during auto-oxidation of NQO1, we used luminol-based chemiluminescence in the presence and absence of HRP (12.5 U/ml). Reactions (0.2 ml) containing 50 mM potassium phosphate buffer, pH 7.4, 200 µM NADH, 80 µg of NQO1, and 0.5 mM luminol were mixed for 1 min at 27°C after which samples were immediately placed into a luminometer. Experiments were repeated with the addition of catalase (177 U/ml) or SOD (10 U/ml).
Spectral Analysis of the Reaction of Superoxide with NQO1. Spectrophotometric analysis was used to monitor the NQO1 catalytic cycle (NADH oxidation and FAD reduction) in NQO1 in the presence and absence of potassium superoxide. Reaction conditions (0.5 ml) were: 40 nmol of recombinant human NQO1, 50 nmol of NADH, and 5 µmol of potassium superoxide. Reactions were performed in 50 mM potassium phosphate buffer, pH 7.4, at 27°C. Scans (250-500 nm) were taken every 40 s. The potassium superoxide stock solution was prepared by adding 1 g of potassium superoxide to 1 ml of anhydrous ME2SO, vigorously mixing for 2 min, followed by centrifugation to remove excess potassium superoxide yielding a stock concentration of approximately 3.6 mM (Reiter et al., 2000).
Oxidation of Dihydroethidium. Oxidation of DHE by was monitored by spectroscopy using a fluorescent compatible microplate reader (Molecular Devices, Sunnyvale, CA). Reactions (1 ml) included 50 mM potassium phosphate buffer, pH 7.4, 0.1 mM diethylenetriaminepentaacetic acid, 0.75 mM xanthine, 0.23 U of XO, and 10 µM dihydroethidium. Reactions were started by the addition of XO and were monitored at excitation and emission wavelengths of 480 and 586 nm, respectively, from 0 to 15 min at 27°C.
ESR Spectroscopy. X-band ESR spectra were recorded at room temperature on a Varian E4 spectrometer (Varian, Inc., Palo Alto, CA) equipped with a TE102 cavity. Typical experimental conditions were: microwave frequency, 9.27 GHz; microwave power, 20 mW; modulation frequency, 100 KHz; modulation amplitude, 0.1 mT; and the applied magnetic field was 300 mT at a scan rate of 8 mT/2 min. Reactions (0.1 ml) were in 50 mM potassium phosphate buffer, pH 7.4, 0.1 mM diethylenetriaminepentaacetic acid, and 25 mM EMPO, and reactions were started by the addition of 1 µl of potassium superoxide dissolved in ME2SO (for stock concentration, see above).
Pyrogallol Auto-Oxidation. Inhibition of pyrogallol auto-oxidation was performed as described previously (Marklund and Marklund, 1974). In brief, 1-ml reactions were monitored for increased absorbance at 420 nm from 0 to 5 min at 27°C. Rates were determined from 3 to 5 min. Complete reactions contained 50 mM Tris-HCL, pH 8.0, 2 mM EDTA, 0.2 mM NADH or NADPH, 12 µg of NQO1, 2.5 µM ES936, and 0.2 mM pyrogallol. In studies with human cell lines, freshly prepared cytosol replaced purified recombinant NQO1.
Kinetic Parameters. Kinetic parameters for the reduction of by NQO1 were obtained using xanthine/XO as the source of and monitoring NQO1-dependent NADH oxidation at 340 nm. Reactions (1 ml) were performed at 27°C in 50 mM potassium phosphate buffer, pH 7.8, 0.5 mM xanthine, and 0.2 mM NADH. In these experiments, we varied the XO activity from 0 to 0.7 units and quantified the rate of production using the reduction of 0.05 mM cytochrome c (monitored at 550-540 nm). We then substituted NQO1 (20-80 µg) in place of cytochrome c and monitored a linear decrease in absorbance at 340 nm (NADH oxidation) for 2 min. The background rate of NADH oxidation was determined at each XO concentration in the absence of NQO1, and this was subtracted from the rate determined in the presence of NQO1. Kinetic parameters (Km and Vmax) were calculated from the rates of NADH oxidation using Sigma Plot and its associated enzyme kinetics module (SPSS Inc., Chicago, IL).
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and/or hydrogen peroxide were formed during the auto-oxidation of NQO1, we used luminol-based chemiluminescence in the presence and absence of HRP (Fig. 1). In the absence of HRP, no chemiluminescence was detected during the auto-oxidation of NQO1, indicating that was not being generated in sufficient quantities to oxidize luminol. The inclusion of HRP, however, resulted in a robust signal that was greater than 95% inhibited by catalase. These data indicate that hydrogen peroxide was the major product formed during the auto-oxidation of NQO1. The inclusion of SOD along with HRP also resulted in a greater than 80% inhibition of the signal, and this can be explained by the ability of SOD to slow the overall rate of NQO1 auto-oxidation (Table 1). In control experiments, no inhibition of NQO1 activity was observed when the purified NQO1 was incubated with hydrogen peroxide (100 mM) for 1 h in the presence or absence of NADH (data not shown), indicating that the inhibition in chemiluminescence was not because of direct inactivation of NQO1 by hydrogen peroxide. The auto-oxidation of NQO1 was also examined by UV-visible spectroscopy. In these experiments, we monitored the spectrum of NADH and FAD following reduction of NQO1 in the presence and absence of potassium superoxide (Fig. 2). These data clearly show that the rate of NADH oxidation is greatly accelerated in the presence of (Fig. 2B); conversely, the addition of SOD significantly inhibited the rate of NADH oxidation (Table 1). No oxidation of NADH by potassium superoxide was detected in the absence of NQO1 (Fig. 2, inset), indicating that these reactions are mediated through NQO1. These data clearly demonstrated an enhanced rate of flavin oxidation in NQO1 in the presence of and suggested that NQO1 may be capable of efficiently catalyzing the reduction of to hydrogen peroxide using reduced pyridine dinucleotide cofactors.
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Superoxide-Dependent Oxidation of Dihydroethidium. The ability of NQO1 to scavenge superoxide was examined using the oxidation of DHE (Fig. 3). In these experiments, the xanthine/XO system was used to generate , which then oxidized DHE to generate a fluorescent product (Zhao et al., 2003). The addition of NADH and NQO1 significantly inhibited the oxidation of DHE by No inhibition was observed when NADH or NQO1 were added separately or when ES936, a mechanism-based inhibitor of NQO1 (Winski et al., 2001; Dehn et al., 2003) was added in combination with NQO1 and NADH. The addition of SOD completely inhibited xanthine/XO-dependent increase in DHE fluorescence, confirming as the primary oxidant of DHE (Fig. 3). Catalase had no effect on xanthine/xanthine oxidase-induced DHE fluorescence, indicating that hydrogen peroxide is not involved in these reactions (Fig. 3). Control experiments demonstrated that NQO1 and NADH could not directly reduce the oxidized product of DHE back to DHE (data not shown). Oxygen consumption studies (Clark electrode) also demonstrated that the combination of NQO1 and NADH had no effect of the rate of xanthine/XO-catalyzed molecular oxygen consumption, indicating that NQO1 and NADH do not effect the rate of production by xanthine/XO (data not shown).
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ESR Spectroscopy Studies. ESR spectroscopy in combination with the spin probe EMPO and potassium superoxide were used to examine the scavenging ability of NQO1 (Fig. 4). In these experiments, the addition of either NQO1 or NADH separately had no effect on signal intensity (Fig. 4, B and C); however, NQO1 in combination with NADH resulted in near complete elimination of the EMPO: adduct signal (Fig. 4D), similar to what was observed for SOD (Fig. 4F). As seen previously, the addition of ES936 reversed the inhibitory effects of NADH/NQO1 (Fig. 4E).
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Auto-Oxidation of Pyrogallol. Inhibition of the auto-oxidation of pyrogallol was used as an additional measure of the ability of NQO1 to scavenge (Fig. 5A). In agreement with previous data, the combination of NQO1 and NADH completely prevented the auto-oxidation of pyrogallol, whereas no inhibition of pyrogallol auto-oxidation was observed with the addition of either NADH or NQO1 separately, Incubation with ES936 blocked the effect of NADH/NQO1 on pyrogallol auto-oxidation. The addition of SOD completely inhibited pyrogallol auto-oxidation, whereas the addition of catalase had no effect demonstrating that is the primary oxidant in this reaction. A linear relationship was observed between inhibition of pyrogallol oxidation and the NQO1 protein concentration (Fig. 5B). Oxygen consumption studies (Clark electrode) demonstrated that the combination of NQO1 and NADH had no effect of the rate of molecular oxygen consumption during the auto-oxidation of pyrogallol (data not shown).
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Kinetic Analysis. Kinetic analysis of the reaction of with NQO1 was performed using xanthine/XO as the source of Kinetic parameters were determined using the NQO1-dependent oxidation of NADH. In these experiments, we added increasing quantities of XO and monitored the effect on the rate of NQO1-dependent NADH oxidation. The rate of production by xanthine/XO was quantified in parallel experiments using the reduction of cytochrome c in the absence of NQO1. Kinetic parameters (Km and Vmax) for the reaction of with NQO1 (Table 2) were calculated from reaction rate versus substrate plots (Fig. 6) fit to the classic Michaelis-Menten equation: V = Vmax x [S]/Km + [S]. From these data, we calculated a rate constant for the reaction of with NQO1 to be 3.60 x 104 M/s and a Km of 2.87 µM at pH 7.8 (Table 2).
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Role of NQO1 in the . Scavenging Activity of Human and Animal Cell Lines. To measure whether the expression of NQO1 influences scavenging in cells, we used isogenic cell lines that differ in the amount of NQO1 protein and activity. The parent CHO cell line (CHO) has no detectable NQO1 activity or protein in cytosolic preparations, whereas the stable clone CHO/812 overexpressing human NQO1 has an activity of greater than 3500 nmol of DCPIP/min/mg of protein. The human breast cancer cell line MDA468 (parental) has a NQO1 activity <5 nmol DCPIP/min/mg of protein, whereas the stable, NQO1 transfected cell line MDA468/NQ16 has a an activity greater than 2500 nmol DCPIP/min/mg of protein. Inhibition of pyrogallol auto-oxidation was used as a measure of scavenging activity (Fig. 7). The addition of NAD(P)H along with cytosol from the CHO/812 or MDA468/NQ16 cell lines resulted in a significant increase in scavenging activity compared with identical samples assayed in the absence of NAD(P)H, and this increase in scavenging activity could be inhibited by ES936. In contrast, no increase in scavenging activity was observed in the presence of NAD(P)H in the NQO1-null CHO and MDA468 cell lines. In the absence of reduced pyridine dinucleotide cofactor, similar scavenging activities were observed between the CHO and CHO/812 lines and MDA and MDA/812 lines, presumably because of equal expression of Cu/Zn SOD. Immunoblot analysis confirmed that overexpression of NQO1 in MDA468/NQ16 cells did not change the protein levels of Cu/Zn SOD compared with the parental MDA468 cell line (data not shown).
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Effect of NQO1 Overexpression on Pyrogallol Cytotoxicity. We examined whether overexpression of NQO1 could increase protection against -induced cytotoxicity. For these assays, we used the parental CHO cell line (no NQO1 activity) and the CHO/812 cell line overexpressing human NQO1 and exposed these cells to increasing concentrations of pyrogallol in HEPES medium (pH 8.0) for 2 h. After treatment, pyrogallol was removed, and the cells were screened for toxicity using the MTT assay. At all pyrogallol concentrations tested, the CHO-DTD812 cells had greater cell survival than the parental CHO cell line (Fig. 8).
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and that the rate of NQO1 auto-oxidation was inhibited by the inclusion of SOD. These results, although novel for NQO1, have been described previously for the auto-oxidation of free flavins and other flavoproteins (Massey et al., 1973; Muller, 1987) and are described in Scheme 1. Under this scheme, the reaction of molecular oxygen with flavinhydroquinone generates a hydroperoxyflavin (I) that decomposes rapidly to flavinsemiquinone radical and (II). The flavinsemiquinone can either react with molecular oxygen to form flavinquinone and (III) or react with to generate flavinquinone and hydrogen peroxide (IV). can also oxidize the flavinhydroquinone to flavinsemiquinone and hydrogen peroxide (V). Experiments by Massey et al. demonstrated that the addition of SOD could significantly decrease the rate of flavinhydroquinone auto-oxidation and concluded that was the primary oxidant responsible for the oxidation of flavinhydroquinone (Massey et al., 1973). Our data also demonstrated that the addition of SOD inhibited NQO1 auto-oxidation by greater than 75% (Table 1). The similarities between the auto-oxidation of free flavin and NQO1 are not surprising given that the FAD cofactor in NQO1 is not covalently bound to the protein. Data from X-ray crystallographic studies with NQO1 have shown that FAD is located within a highly plastic active site and held in place through hydrogen bonding and van der Waals interactions (Faig et al., 2000). An important feature of NQO1-bound FAD is that the protein environment surrounding FAD does not stabilize the flavinsemiquinone (Tedeschi et al., 1995a, 1995b). Whether interacts with other flavoproteins with comparably similar flavin redox chemistry to NQO1 is unknown. The ability of NQO1 bound FAD to carry out the reduction of in reactions similar to those described for the auto-oxidation of free flavin (IV and V) in combination with the ability of NQO1 to rapidly regenerate the flavinhydroquinone from flavinquinone via the oxidation of NAD(P)H suggested to us that NQO1 may have an embedded scavenging activity (Scheme 2).
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In assays designed to test the scavenging activity of NQO1, we confirmed that NQO1 in combination with NAD(P)H could effectively inhibit a wide range of -dependent reactions. In addition, we demonstrated that the scavenging activity of NQO1 could be inhibited by ES936, a mechanism-based inhibitor of NQO1. These results are important because they demonstrate that the scavenging activity of NQO1 is dependent on the catalytic turnover of the enzyme and not because of an unknown contaminant. Kinetic analysis of the reaction of with NQO1 demonstrated that -dependent oxidation of NADH by NQO1 is a saturable process with increasing concentration and that reduction is the rate-limiting step in this reaction. The fact that this reaction is saturable and follows classic Michaelis-Menten kinetics differentiates this mechanism of enzymatic detoxification from that of SOD, which is not saturable up to the experimental limits of concentration (Bull and Fee, 1985). The relationship between NQO1 and SOD in their kinetics of inactivation with has some similarities between the relationship between catalase (high-capacity, high Km) and glutathione peroxidase (low-capacity, low Km) for breakdown of hydrogen peroxide (Makino et al., 1994; Sasaki et al., 1998).
The primary metabolic function of NQO1 is considered to be the reduction of quinones to hydroquinones. An embedded scavenging activity of NQO1 is attractive because it would serve a dual purpose. Any generated by auto-oxidation of hydroquinones would be scavenged at its site of generation. Second, because is the major driving force for hydroquinone oxidation (Ollinger et al., 1990), removal would prevent the acceleration of HQ autoxidation helping to preserve the molecule in the hydroquinone form and facilitating excretion.
Aside from a potential role in minimizing the deleterious consequences of the generation of redox active hydroquinones, a superoxide scavenging role for NQO1 raises the issue of the relevance of this effect in cells where there are more efficient systems for superoxide removal such as SOD. The kinetic parameters obtained for the reaction of with NQO1 indicate that the Kcat/Km ratio is 3.6 x 104 M/s, and the Km is 2.87 µM. The rate of chemical dismutation of at pH 7.8 was approximated from data in a previous study (McClune and Fee, 1976). Although performed under different conditions, the rate of chemical dismutation was in the order of 2.5 x 104 M/s. The kinetic constants obtained in our studies for the rate of the reaction of with NQO1 may be artificially low because xanthine, used as a component of the generating system, was shown to inhibit NQO1 activity by 20% (data not shown; also see Roberts et al., 1989). A more accurate measurement of the reaction kinetics for the interaction of with NQO1 by stopped-flow methodologies using authentic is warranted. Nevertheless, the kinetic data suggest that the rate of reaction of with NQO1 is less than an order of magnitude higher than the rate of chemical dismutation of and is in the order of 4 orders of magnitude less than the rate of enzymatic dismutation catalyzed by SOD (1.69 x 109 M/s). It should be borne in mind, however, that the significance of NQO1 as a cellular scavenger of superoxide will not depend solely upon the reaction rate but also on the concentration of NQO1 in cells relative to other scavenging systems such as SOD. NQO1 is highly inducible in cells after exposure to oxidative stress, and its extensive and rapid induction would be consistent with a role for NQO1 as part of a stress or adaptive response against oxygen radicals such as superoxide. We approached the cellular relevance of the reaction of NQO1 and using isogenic cell lines differing only in NQO1 levels. The parental CHO cell line has no NQO1 activity or protein, whereas MDA468 cells have only trace levels of NQO1 because of a homozygous genetic polymorphism (NQO1*2/*2) and were stably transfected with human NQO1 to generate CHO/812 and MDA468NQ16 cell lines that contain very high levels of NQO1 activity. Our data demonstrated that expression of high levels of NQO1 in cell cytosols resulted in increased scavenging of , and the specificity of this reaction for NQO1 was confirmed using ES936, a mechanism-based inhibitor of NQO1. In addition, experiments in CHO cells demonstrated that overexpression of NQO1 resulted in protection against pyrogallol-induced toxicity. These data suggest that high levels of NQO1 protein may compensate for the slower rate of reaction of with NQO1 compared with SOD. High levels of NQO1 have been detected by immunohistochemistry in normal human tissues naturally exposed to sustained levels of molecular oxygen such as cornea and lens epithelium, vascular and alveolar endothelium, and bronchial epithelium (Siegel et al., 1998; Siegel and Ross, 2000). Based on these data, we suggest that NQO1 may function as an additional enzyme system to SOD for scavenging of in cellular systems, and this effect may be particularly marked in cells containing high levels of NQO1.
Consistent with a role for NQO1 in scavenging , it was shown very recently that treatment of a pancreatic cancer cell line with the NQO1 inhibitor dicumarol increased intracellular production and inhibited cell growth and the in vitro malignant phenotype via a superoxide-dependent mechanism (Cullen et al., 2003).
It is conceivable that the absence of NQO1 may result in diminished protection against over the lifetime of an individual, leading to increased cell damage and incidence of disease. This is especially relevant to NQO1 because of a common single nucleotide polymorphism (NQO1*2). Expression of the NQO1*2 allele results in a NQO1 protein that has minimal catalytic activity and is rapidly degraded by the ubiquitin/proteasomal system (Traver et al., 1997; Siegel et al., 2001). Individuals with one variant allele have diminished NQO1 activity and protein levels, whereas individuals with two variants alleles have only trace or nondetectable levels of NQO1 protein and activity (Siegel et al., 1999). Increased rates of many cancers including lung, colon, skin, bladder, and leukemias have been found to correlate with the NQO1*2 allele frequency (Schulz et al., 1997; Clairmont et al., 1999; Larson et al., 1999; Lafuente et al., 2000; Lewis et al., 2001). The incidence of other diseases such as Parkinson's disease has also been found to correlate with NQO1*2 allele frequency (Harada et al., 2001).
In summary, we have demonstrated that NQO1 posses NAD(P)H-dependent scavenging activity. The reaction of. and NQO1 is mediated through reduced forms of FAD that can rapidly be regenerated via the oxidation NAD(P)H. The ability of NQO1 to scavenge may be one reason for the rapid induction of this enzyme following diverse forms of oxidative stress.
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Footnotes |
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ABBREVIATIONS: NQO1, NAD(P)H:quinone oxidoreductase 1; DHE, dihydroethidium; DCPIP, 2,6-dichlorophenol-indophenol; MTT, 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide; XO, xanthine oxidase; SOD, superoxide dismutase; HRP, horseradish peroxidase; EMPO, 2-ethoxycarbonyl-2-methyl-3,4-dihydro-2H-pyrrole-1-oxide; ES936, 5-methoxy-1,2-dimethyl-3-[(4-nitrophenoxy)methyl]indole-4,7-dione; CHO, Chinese hamster ovary; ESR, electron spin resonance.
Address correspondence to: Dr. David Ross, Department of Pharmaceutical Sciences, School of Pharmacy, University of Colorado Health Sciences Center, 4200 East 9th Ave., Denver, CO 80262. E-mail: david.ross{at}uchsc.edu
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, xanthine/XO/dihydroethidium; 
, plus NADH (0.2 mM); 
, plus NQO1 (25 µg/ml); 
, plus NADH (0.2 mM) and NQO1 (25 µg/ml); 
, plus NADH (0.2 mM), NQO1 (25 µg/ml), and ES936 (4 µM); 
, plus superoxide dismutase (5 U/ml); 
, plus catalase (100 U/ml). Data are representative of five separate experiments.
