Introduction

Summary Introduction Materials & Methods Results Discussion References

NQO1 (EC 1.6.99.2), or DT-diaphorase, is an obligate two-electron reductase that catalyzes reduction of a broad range of substrates (1, 2). It is a flavoprotein that exists as a homodimer and is biochemically characterized by its unique ability to utilize either NADH or NADPH as reducing cofactors and by its inhibition by the anticoagulant dicumarol (1, 3). The enzyme is generally considered as a detoxification enzyme because of its ability to detoxify reactive quinones and quinone-imines to less reactive and less toxic hydroquinones (1, 3). Such a two-electron reduction also bypasses semiquinone production and thus prevents the generation of reactive oxygen species derived from interaction of the semiquinone with molecular oxygen (4, 5). The ability of NQO1 to deactivate many reactive species, including quinones, quinone-imines, and azo compounds, demonstrates its importance as a chemoprotective enzyme (6-10). The other major protective effect of NQO1 is to function as a cancer preventive enzyme (6-9), which has been recognized for >30 years (11).

Whether NQO1 catalyzes the reduction of endogenous substrates remains unclear. A potential role for NQO1 in vitamin K metabolism has been suggested (12, 13), but studies with purified NQO1 do not support such a role (14). An important observation is that x-radiation and UV radiation, which are known to generate oxidative stress, induced NQO1 expression >30-fold in human cells (15). Our data show that hepatic loading of iron in rats generates oxidative stress and a concomitant induction of NQO1.¹ This suggests that in addition to limiting oxygen radical formation from exogenous quinones, NQO1 may play an endogenous antioxidant role. Recent work has suggested that NQO1 maintains ubiquinone (CoQ₁₀) in its quinol form, which can act as an antioxidant to protect membranes from oxidative stress (16).

We examined the role of NQO1 in the metabolism of -tocopherol derivatives. In this report, we demonstrate that TQ is an excellent substrate for human NQO1. TQ is produced via free radical attack of -tocopherol (vitamin E) and has no intrinsic antioxidant activity (17-19). The product of NQO1-mediated reduction of TQ is TQH₂ which, unlike TQ, is a potent antioxidant (18, 19). Reduction of TQ to its TQH₂ derivative has been demonstrated in cellular systems, but the enzymes responsible have not been characterized (18-20). This work demonstrates a role for NQO1 in -tocopherol metabolism and suggests that one of the physiological functions of NQO1 may be to regenerate antioxidant forms of -tocopherol.

	Materials and Methods

Summary Introduction Materials & Methods Results Discussion References

Chemicals. NADH, NADPH, cholic acid, dicumarol, DCPIP, arachidonic acid, butylated hydroxytoluene, -tocopherol acetate, cumene hydroperoxide, 2-thiobarbituric acid, HEPES, and CoQ₁₀ were purchased from Sigma Chemical (St. Louis, MO). TQ was obtained from United States Biochemical (Arlington Heights, IL). Stock solutions of TQ were prepared in 95% (v/v) ethanol unless otherwise stated. All other reagents were of analytical grade.

CHO cell lines and NQO1 purification. CHO cell lines stably transfected with human NQO1 cDNA were developed as previously described (21). CHO cell lines were grown in Ham's F-12 (GIBCO BRL, Gaithersburg, MD) supplemented with 10% (v/v) fetal bovine serum and 2 mM glutamine, 100 units/ml penicillin, and 100 µg/ml streptomycin (GIBCO BRL). Recombinant human NQO1 protein was purified from Escherichia coli using Cibacron Blue affinity chromatography as previously described (22). The specific activity of the purified NQO1 protein was 625 µmol of DCPIP/min/mg. NQO1 specific activity in the CHO cell lines and purified NQO1 protein was determined using DCPIP reduction as previously described (23). Protein concentrations were determined according to the method of Lowry (24).

Formation of TQH₂ in CHO sonicates and cultured cells. The reduction of TQ by CHO sonicates was performed by the addition of 650 µg of CHO sonicate to 50 mM potassium phosphate buffer, pH 7.4, containing 1% (w/v) cholic acid, 200 µM NADH, and 50 µM TQ (final volume, 250 µl) at 27°. After 30 min, the reactions were terminated, and TQ and TQH₂ analyses were performed as described below. The conversion of TQ to TQH₂ in the cultured CHO cell lines was determined using the following methods: CHO cell lines were grown to >90% confluency, the medium was removed, and fresh medium containing 50 µM TQ was added. After 6, 12, or 24 hr, the reaction was terminated by removal of the TQ-containing medium, washing of the cells with 10 ml of PBS twice, and scraping of the cells into 1 ml of PBS. The cells were pelleted by centrifugation, the supernatant was removed, and the cell pellet was snap-frozen in liquid nitrogen and stored at 70° until analysis by HPLC.

Analysis of TQ and TQH₂ HPLC was used to separate TQ and TQH₂ in reactions with either purified NQO1, CHO sonicates or CHO cell lines. Reactions (purified NQO1, CHO sonicates) were stopped with an equal volume of cold acetonitrile containing 0.01% (w/v) butylated hydroxytoluene and 200 µM -tocopherol acetate (internal standard) and then centrifuged at 10,000 rpm for 2 min. The cell pellet (see above) was resuspended in 150 µl of acetonitrile containing 0.003% (w/v) butylated hydroxytoluene and 67 µM -tocopherol acetate and then centrifuged at 10,000 rpm for 2 min. The supernatant was analyzed on a LiChrosorb RP-18 column (5 µm, 25 cm, Merck, Darmstadt, Germany) with the following gradient: buffer A, 1% (v/v) acetic acid; buffer B, 100% acetonitrile; gradient, 70-95% buffer B over 10 min and then hold at 95% buffer B for 25 min. Flow rate was maintained at 1.5 ml/min with UV detection at 280 nm. HPLC retention times were TQH₂, 14 min; TQ, 22.1 min; and internal standard, 33.3 min. TQH₂ was analyzed by capillary GC-MS on a Fisons MD800 instrument (Fisons Instruments, Beverly, MA) equipped with a Carlo Erba 8000 series gas chromatograph and a Fisons on-column injector and operated in the electron ionization mode at 70 eV. The HPLC peak corresponding to TQH₂ was collected, TQH₂ was extracted from the mobile phase with hexane, and the hexane was evaporated in vacuo. TQH₂ was converted to the tris-(O-trimethylsilyl) derivative as described (25). The derivative was analyzed by GC-MS on a 30 m × 0.25 mm DB-5ms column (J & W Scientific, Folsom, CA) with cold on-column injection (25). The tris-(O-trimethylsilyl) derivative of TQH₂ coeluted with an authentic standard and yielded a mass spectrum identical to that of the standard: m/z 665 [M+, 16%] 575 [21], 341 [8], 309 [100], 294 [20], 280 [10], 236 [9], 220 [6]. This spectrum is essentially identical to that we previously reported for TQH₂ (25). The molecular ion for the tris-(O-trimethylsilyl) derivative appeared at m/z 665 due to mass defect; the monoisotopic mass is 664.5 amu.

Kinetic analysis of TQ and CoQ₁₀ reduction by NQO1. Kinetic analysis of TQ reduction by NQO1 was determined by spectrophotometrically monitoring NADH oxidation. Reactions (1 ml) were performed in 50 mM potassium phosphate buffer, pH 7.4, containing 5% (w/v) cholic acid, 0.4% (v/v) Tween-80, 200 µM NADH, 0.288 µg of NQO1, and 0.01-1.28 mM TQ. Reactions were monitored for 0-2 min at 27° at 340 nm. Kinetic parameters were determined using Enzfitter kinetic software (Biosoft, Cambridge, UK) from the mean of triplicate determinations. A comparison of the rate of reduction of TQ and CoQ₁₀ (5, 50 µM) by NQO1 was determined by monitoring NADH oxidation as described above. Reactions (1 ml) were performed in 50 mM potassium phosphate buffer, pH 7.4, containing 5% cholic acid and 0.4% Tween-80, 200 µM NADH, and 7.2 µg of NQO1. For these experiments, initial stock solutions of TQ and CoQ₁₀ (10 mM) were dissolved in N,N-dimethylformamide before dilution in buffer.

Lipid peroxidation in rat liver microsomes and CHO cells. Lipid peroxidation was measured by analysis of TBARS as previously described (26). Reactions with rat liver microsomes were performed as follows: 200 µM NADH, 3.6 µg of NQO1, 50 µM TQ, and 750 µg of microsomes were added to 50 mM potassium phosphate buffer, pH 7.4, containing 1% cholic acid (final volume, 0.5 ml) at 27°. After 30 min, cumene hydroperoxide (1 mM) was added to initiate lipid peroxidation. After 1 hr, the reaction was stopped by the addition of 100 µM butylated hydroxytoluene and 2 ml of 0.25 N hydrochloric acid containing 15% (w/v) trichloroacetic acid and 0.37% (w/v) 2-thiobarbituric acid. The sample was heated to 80° for 20 min and then centrifuged at 2500 rpm for 10 min, and the absorbance at 535 nm was determined. Results were expressed as TBARS equivalents using a molar extinction coefficient of 1.56 × 10⁵ (26). Lipid peroxidation studies with CHO cell lines were performed as follows: CHO cell lines were grown to >90% confluency on 100-mm tissue culture plates. Before the initiation of lipid peroxidation, the medium was exchanged with fresh medium containing 250 µM arachidonic acid and 0, 1, or 5 µM TQ. After 14 hr, the TQ-containing medium was removed, the cells were washed with 10 ml of PBS, and 10 ml of Krebs-HEPES buffer, pH 7.2, was added. Lipid peroxidation was initiated by the addition of cumene hydroperoxide (1 mM), and after 2.5 hr, the cells were scraped into the medium and collected by centrifugation. The supernatant was removed, and the cell pellet was resuspended in 1 ml of PBS. An aliquot (10 µl) of cell suspension was removed for protein determination according to the method of Lowry (24), and lipid peroxidation was measured using the TBARS assay in the remaining cell suspension as described above.

Statistical analysis. Results are expressed as mean ± standard deviation. Statistical significance between sets of data was determined using the Student's t test.

	Results

Summary Introduction Materials & Methods Results Discussion References

TQ as a substrate for purified NQO1. Initial experiments were performed to determine whether TQ could serve as a substrate for purified NQO1. HPLC analysis of a reaction containing TQ, NADH, and NQO1 showed loss of the TQ peak (t_R = 22.1 min) and formation of a more polar product (t_R = 14 min; Fig. 1A). The product from the reduction of TQ by NQO1 was confirmed by GC-MS as TQH₂ (for details, see Materials and Methods). The reaction was NADH or NADPH dependent and dicumarol inhibitable (Fig. 1B). Kinetic values for the reduction of TQ by NQO1 were determined spectrophotometrically by monitoring the oxidation of NADH. Values for K_m, k_cat, and k_cat/K_m are presented in Table 1 and suggest that NQO1 efficiently reduces TQ to TQH₂. Kinetic parameters for the reduction of CoQ₁₀ by NQO1 could not be determined due to the poor solubility of CoQ₁₀. Comparative studies on the reduction of TQ and CoQ₁₀ by NQO1 were performed at substrate concentrations of 5 and 50 µM (Table 2). The rate of TQ reduction was ~36-fold greater at 5 µM and ~57-fold greater at 50 µM compared with the rate of CoQ₁₀ reduction. These data demonstrate that NQO1 can reduce TQ considerably more efficiently than CoQ₁₀.

View larger version (22K): [in this window] [in a new window]   Fig. 1.   HPLC analysis of the reduction of TQ by purified NQO1. Reaction conditions were 50 µM TQ, 200 µM NADH, and 7.2 µg of NQO1 in 50 mM potassium phosphate buffer, pH 7.4, containing 1% cholic acid. Reactions were performed in a total volume of 1 ml at 27° for 30 min in the absence (A) and presence (B) of 20 µM dicumarol. HPLC conditions are described in Materials and Methods.

Reduction of TQ by NQO1-transfected CHO cell sonicates. The reduction of TQ by sonicates prepared from CHO cell lines stably transfected with NQO1 and expressing different amounts of NQO1 activity was examined by HPLC (Table 3). A relationship between TQ reduction and NQO1 activity was apparent. Sonicates prepared from the CHO cell line with the highest NQO1 activity (CHO₈₁₂) catalyzed the greatest amount of TQ reduction. Less TQ reduction was observed in sonicates from the CHO cell line CHO₈₁₅, which contains intermediate levels of NQO1, whereas sonicates from the NQO1-deficient CHO parent cell line (CHO_glyA) catalyzed only minimal TQ reduction. The reduction of TQ by the CHO₈₁₂ and CHO₈₁₅ sonicates was NADH or NADPH dependent and dicumarol inhibitable. The product of the reduction of TQ by CHO sonicates cochromatographed with TQH₂ prepared from the reduction of TQ by purified NQO1 (data not shown).

Reduction of TQ by NQO1-transfected CHO cells in culture. Experiments were performed to examine TQ reduction and TQH₂ formation in NQO1-transfected CHO cells in culture (Fig. 2). Previous studies have shown that there were no significant differences between the CHO₈₁₂ and CHO_glyA cell lines in the levels of one-electron reductases; NADPH:cytochrome P450 reductase, and NADH:cytochrome b₅ reductase (21). The NQO1-rich CHO₈₁₂ and NQO1-deficient CHO_glyA cell lines were exposed to 50 µM TQ in culture, and the amounts of TQ and TQH₂ were determined by HPLC after 6 and 12 hr. The results are expressed as the ratio of TQH₂ to TQ and show that the high-NQO1 cell line, CHO₈₁₂, had significantly higher TQH₂-to-TQ ratios compared with the NQO1-deficient cell line, CHO_glyA. These data indicate that cells with high NQO1 can generate and maintain higher levels of TQH₂ than NQO1-deficient cells.

View larger version (34K): [in this window] [in a new window]   Fig. 2.   The formation of TQH2 in NQO1-transfected CHO cell lines. CHOGlyA and CHO812 cell lines were treated with 50 µM TQ in culture; at 6 and 12 hr, the cells were harvested, and the amounts of TQH2 and TQ were determined by HPLC. Data represent mean ± standard deviation values from three separate determinations (, CHOGlyA; , CHO812). *, CHO812 cells were significantly different from CHOGlyA cells (p < 0.05).

Antioxidant actions of TQH₂. Experiments were performed to determine the stability of TQH₂. HPLC analysis confirmed that TQH₂ formed in the reaction with NQO1 and stoichiometric amounts (50 µM) of TQ and NADH was stable under aerobic conditions. No autoxidation of TQH₂ back to TQ could be detected after 3 hr in 50 mM potassium phosphate buffer, pH 7.4, containing 1% cholic acid. The oxidation of TQH₂ to TQ, however, could readily be induced by unsaturated fatty acid peroxidation (Fig. 3A). In reactions in which TQH₂ was formed after the reduction of TQ by NQO1, the addition of arachidonic acid, Fe²⁺, and hydrogen peroxide catalyzed the oxidation of TQH₂ back to TQ. However, when arachidonic acid was absent or replaced by stearic acid, a saturated fatty acid, minimal oxidation of TQH₂ to TQ could be observed (Fig. 3, B and C). These data suggest that TQH₂ can be oxidized back to TQ via products of unsaturated fatty acid peroxidation.

View larger version (29K): [in this window] [in a new window]   Fig. 3.   HPLC analysis of the oxidation of TQH2 by arachidonic acid peroxidation. Reaction conditions were 50 µM TQ, 50 µM NADH, and 7.2 µg of NQO1 in 50 mM potassium phosphate buffer, pH 7.4, containing 1% cholic acid. After 30 min, we added (A) 5 mM arachidonic acid, 50 µM ferrous sulfate, and 500 µM hydrogen peroxide; (B) 50 µM ferrous sulfate and 500 µM hydrogen peroxide; and (C) 5 mM stearic acid, 50 µM ferrous sulfate, and 500 µM hydrogen peroxide, and the reactions were allowed to proceed for 90 min. HPLC conditions are described in Materials and Methods.

View larger version (41K): [in this window] [in a new window]   Fig. 4.   Inhibition of cumene hydroperoxide-induced lipid peroxidation in NQO1-transfected CHO cell lines by pretreatment with TQ. CHOGlyA and CHO812 cell lines were pretreated with 250 µM arachidonic acid and 0, 1, or 5 µM TQ and then exposed to 1 mM cumene hydroperoxide in culture. Reaction conditions are described in Materials and Methods. Data represents three separate determinations (± standard deviation: , CHOGlyA; , CHO812). *, CHO812 cells were significantly different from CHOGlyA cells (p < 0.05).

	Discussion

Summary Introduction Materials & Methods Results Discussion References

The data presented in this report indicate that TQ can be reduced to TQH₂ in cell-free and cellular systems by NQO1 and that TQH₂ can function as an effective antioxidant. Previous work has shown that TQ can be metabolized to TQH₂ by rat liver microsomal, mitochondrial, and cytosolic preparations (18-20). The reduction of TQ to TQH₂ in these preparations was either NADPH or NADH dependent, which suggests that many cellular reductases are capable of catalyzing this reaction. There are limited data available that characterize the enzymes responsible for the reduction of TQ to TQH₂. One study has shown that purified NADPH-cytochrome P450 reductase can reduce TQ to TQH₂ (20), and a second study has suggested that TQ is a substrate for purified human brain carbonyl reductase (29).

Previous experiments using rat liver microsomes and purified NADPH cytochrome P450 reductase have suggested that TQH₂ is unstable under aerobic conditions, although paradoxically the authors also demonstrated that TQH₂ could be detected after aerobic incubations of isolated hepatocytes with TQ (20). In our experiments, reduction of TQ to TQH₂ by NQO1 proceeded readily under aerobic conditions, and the TQH₂ that was formed was stable with no significant autoxidation detected after 3 hr of incubation. The requirement for anaerobic conditions to detect significant quantities of TQH₂ after metabolism of TQ in previous studies using rat liver microsomes and purified cytochrome P450 reductase probably reflects the instability of the -tocopherol semiquinone radical to oxygen. Indeed, we have found that incubation of rat liver microsomes with NADPH and TQ to generate the -tocopherol semiquinone radical results in extensive oxygen uptake under aerobic conditions (data not shown). Because NQO1 is a two-electron reductase, it can directly form TQH₂ without the intermediacy of the -tocopherol semiquinone radical.

The stability of TQH₂ is important because the hydroquinone form of TQ is responsible for the antioxidant function (18, 19). The current data show that TQH₂ is stable and does not undergo substantial autoxidation. However, the addition of an unsaturated fatty acid (arachidonic acid), Fe²⁺, and hydrogen peroxide did catalyze oxidation of TQH₂ back to TQ. These data indicate that TQH₂, like -tocopherol, is reactive primarily toward lipid-derived radicals. In a study comparing the inhibition of ascorbate/Fe²⁺-induced lipid peroxidation in liposomes, TQH₂ was 5-fold more effective than -tocopherol (18). The ability of a cell to maintain TQ in its reduced form will result in substantial antioxidant protection. TQ pretreatment has been shown to protect cultured cells from lipid peroxidation and cytotoxicity, presumably due to its reduction to TQH₂ (30-33). Our data demonstrate that cells with high NQO1 expression had higher levels of TQH₂ and were more resistant to lipid peroxidation than were cells lacking NQO1. The ability of cells to reduce TQ to TQH₂ via NQO1 therefore represents an effective protective mechanism against lipid peroxidative injury.

Beyer et al. (16) have shown recently that purified rat NQO1 is capable of reducing coenzyme Q derivatives to their quinol forms and that these reduced compounds have substantial antioxidant properties. In their study, the quinol forms of coenzyme Q (CoQ₉, CoQ₁₀) were shown to protect multilamellar vesicles against lipid peroxidation and isolated hepatocytes against Adriamycin-induced oxidative stress. We extended this work from rat to human NQO1 and compared the rate of reduction of CoQ₁₀ and TQ. Under the conditions used in our study, the rate of TQ reduction by NQO1 was markedly greater (36-57-fold) than that observed with CoQ₁₀. The results obtained in both our work and that of Beyer et al. (16) clearly demonstrate that NQO1 can maintain endogenous quinones in a reduced antioxidative state.

It is conceivable that one of the functions of NQO1 may be to regenerate antioxidant forms of -tocopherol (Fig. 5). A role for NQO1 in maintaining physiological levels of -tocopherol from the reduction of -tocopherones by NQO1 has previously been postulated (34). Oxidation of -tocopherol by peroxyl radicals yields 8a-(alkyldioxy)tocopherones, which may either hydrolyze to TQ or be reduced to regenerate -tocopherol (35). The regeneration of -tocopherol from -tocopherones occurs via a two-electron reduction, which could conceivably be catalyzed by a reductase such as NQO1 because this enzyme can catalyze the direct two-electron reduction of a broad range of substrates. Experiments are under way to examine the potential role of NQO1 in the reduction of -tocopherones. In addition, the conversion of TQ into -tocopherol in humans has recently been demonstrated (36); the authors suggest that -tocopherol is regenerated from TQ after enzymatic reduction to TQH₂, although the enzyme(s) responsible have not yet been identified.

View larger version (12K): [in this window] [in a new window]   Fig. 5.   Role of NQO1 in the regeneration of antioxidant forms of -tocopherol (-T). -T·, -tocopherol radical; -TQ, -tocopherolquinone; -TQH2, -tocopherolhydroquinone.

Recent work in our laboratory has shown that NQO1 expression is polymorphic due to an A-T substitution in exon 6 of the NQO1 gene (37, 38). This base pair substitution leads to a proline-to-serine change in the amino acid structure, which results in a total lack of NQO1 protein expression in individuals homozygous for this mutation (37, 38). The polymorphism demonstrates mendelian transmission (39) and occurs with a prevalence of ~6% in whites and ~18% in the Chinese (40). Our data suggest that individuals lacking expression of NQO1 may have a decreased capacity to protect against cellular oxidative damage, and this may have implications for chemoprotection and chemoprevention.

In summary, NQO1 has classically been considered a detoxification enzyme because of its role in the reduction of exogenous quinone substrates to their hydroquinone forms, thus bypassing reactive semiquinone radical formation. The ability of NQO1 to efficiently reduce TQ to TQH₂, however, suggests that one of the physiological functions of NQO1 may be to regenerate antioxidant forms of -tocopherol.