Effect of dicumarol,a Nad(P)h: quinone acceptor oxidoreductase 1 (DT-diaphorase) inhibitor on ubiquinone redox cycling in cultured rat hepatocytes

Ubiquinol is considered to serve as an endogenous antioxidant. However, the mechanism by which the redox state of intracellular ubiquinone (UQ) is maintained is not well established. The effect of dicumarol, an inhibitor of NAD(P)H: quinone acceptor oxidoreductase 1 (NQO1=DT-diaphorase, EC 1.6.99.2), on the reduction of UQ in cultured rat hepatocytes was investigated in order to clarify whether or not NQO1 is involved in reducing intracellular UQ. A concentration of 5 μM dicumarol, which does not inhibit cytosolic NADPH-dependent UQ reductase in vitro , was observed to almost completely inhibit NQO1 and thereby to stimulate cytotoxicity of 2-methyl-1,4-naphthoquinone (menadione) in cultured rat hepatocytes. However, 5 μM dicumarol did not inhibit reduction of endogenous UQ-9, as well as exogenous UQ-10 added to the hepatocytes. In addition, it did not stimulate the formation of thiobarbituric acid reactive substances (TBARS) in the hepatocytes. These results suggested that NQO1 is not involved in maintaining UQ in the reduced state in the intact liver cells.     

Ubiquinone redox cycle as a cellular antioxidant defense system

Ubiquinone (UQ) reductase responsible for reduction of non-mitochondrial UQ was investigated in rats toward demonstrating an antioxidant role of UQ. In the liver, most of cellular UQ-10 reductase activity was attributable to a novel NADPH-UQ reductase in cytosol. The enzyme was not inhibited by dicumarol and rotenone, and had a Km of 19 microM for NADPH and 307 microM for NADH at the optimum pH 7.4. The enzyme was purified 300-fold to apparent homogeneity from the liver cytosol by an affinity chromatographic method. The purified enzyme reduced UQ-10 in lecithin liposomes, and protected the liposomes from lipid peroxidation. Furthermore, supplementation of rats with UQ-10 was observed to increase the enzyme level in their livers without affecting levels of other antioxidant factors. The observations suggested that cytosolic NADPH-UQ reductase is responsible for cellular UQ redox cycle as an endogenous antioxidant.     

Coenzyme Q Cytoprotective Mechanisms for Mitochondrial Complex I Cytopathies Involves NAD(P)H: Quinone Oxidoreductase 1(NQO1)

The commonest mitochondrial diseases are probably those impairing the function of complex I of the respiratory electron transport chain. Such complex I impairment may contribute to various neurodegenerative disorders e.g. Parkinson's disease. In the following, using hepatocytes as a model cell, we have shown for the first time that the cytotoxicity caused by complex I inhibition by rotenone but not that caused by complex III inhibition by antimycin can be prevented by coenzyme Q (CoQ 1 ) or menadione. Furthermore, complex I inhibitor cytotoxicity was associated with the collapse of the mitochondrial membrane potential and reactive oxygen species (ROS) formation. ROS scavengers or inhibitors of the mitochondrial permeability transition prevented cytotoxicity. The CoQ 1 cytoprotective mechanism required CoQ 1 reduction by DT-diaphorase (NQO 1 ). Furthermore, the mitochondrial membrane potential and ATP levels were restored at low CoQ 1 concentrations (5 &#119 M). This suggests that the CoQ 1 H 2 formed by NQO 1 reduced complex III and acted as an electron bypass of the rotenone block. However cytoprotection still occurred at higher CoQ 1 concentrations (>10 &#119 M), which were less effective at restoring ATP levels but readily restored the cellular cytosolic redox potential (i.e. lactate: pyruvate ratio) and prevented ROS formation. This suggests that CoQ 1 or menadione cytoprotection also involves the NQO 1 catalysed reoxidation of NADH that accumulates as a result of complex I inhibition. The CoQ 1 H 2 formed would then also act as a ROS scavenger.     

Cytosolic NADPH-UQ reductase, the enzyme responsible for cellular ubiquinone redox cycle as an endogenous antioxidant in the rat liver.

Cellular ubiquinone (UQ) is expected to act as an endogenous antioxidant against oxidative stress. To confirm this, UQ-reductases which are necessary to regenerate ubiquinol (UQH2) were investigated in rat tissue, and a novel NADPH-dependent UQ (NADPH-UQ) reductase was found in cytosol. The cytosolic NADPH-UQ reductase activity accounted for more than 80% of UQ-10 reduction by the rat liver homogenate in the presence of NADPH. Furthermore, the NADPH-UQ reductase activities in various tissues were correlated to the redox states of UQ in the corresponding tissues. Rat liver cytosol with NADPH protected lecithin liposomes containing UQ-10, as well as UQH2-10 from AMVN (2,2'-azobis(2,4-dimethylvaleronitrile))-induced lipid peroxidation. The enzyme purified from rat liver cytosol, reduced UQ-10 in lecithin liposomes at approximately the same rate as did cytosol. These results supported that cytosolic NADPH-UQ reductase is the enzyme responsible for nonmitochondrial UQ reduction acting as an endogenous antioxidant against oxidative stress. The antioxidant role of the UQ redox cycle and NADPH-UQ reductase was discussed in relation to other cellular NADPH-dependent antioxidant enzymes.     

Hepatocyte metabolism of coenzyme Q1 (ubiquinone-5) to its sulfate conjugate decreases its antioxidant activity

Previous studies on the metabolism of coenyzme Q (CoQ) have focused on products found in the urine, bile or feces. However, the metabolites found in these samples were end products from a multitude of catabolic processes which did not necessarily reflect CoQ intracellular metabolism (e.g. in the liver, the major site of CoQ synthesis or metabolism). Using isolated rat hepatocytes, we have found that the sulfation of coenzyme Q1 (CoQ1) was the initial and dominant step following its reduction to the hydroquinone. This metabolic process is important as conjugation may occur on the hydroquinone metabolites of any coenzyme10 scission product retaining the quinone ring. By using rat liver cytosol, we were able to identify the monosulfated metabolite of CoQ1. The CoQ1 sulfate conjugate was identified by mass spectrometry followed by tandem mass spectrometry. The rate of formation of the CoQ1 sulfate conjugate was markedly increased by the addition of NADH and was prevented by dicumarol, a DT-diaphorase (NQO1) inhibitor. CoQ1 sulfate conjugate formation catalysed by cytosol was inhibited by the sulfotransferase 1A (SULT1A) inhibitor, pentachlorophenol (PCP) suggesting that sulfation was carried out by the SULT 1A isoform. CoQ1 sulfation in isolated hepatocytes and inversely CoQ1 hydroquinone formation were dependent on the concentration of inorganic sulfate in the media. Intracellular sulfation also decreased CoQ1 antioxidant and cytoprotective activity towards cumene hydroperoxide (CHP) induced cell death. Sulfotransferases may therefore play a significant role in endogenous CoQ metabolism following its degradation to short chain products.     

Ubiquinone-10 production using Agrobacterium tumefaciens dps gene in Escherichia coli by coexpression system

Ubiquinone (Coenzyme Q; abbreviation, UQ) acts as a mobile component of the respiratory chain by playing an essential role in the electron transport system, and has been widely used in pharmaceuticals. The biosynthesis of UQ involves 10 sequential reactions brought about by various enzymes. In this study we have cloned, expressed the decaprenyl diphosphate synthase, designated dps gene, from Agrobacterium tumefaciens, and succeeded in detecting UQ-10 in addition to innate UQ-8 in Escherichia coli. Furthermore, the production of UQ-10 was higher than UQ-8. To establish an efficient expression system for UQ-10 production, we used genes, including ubiC, ubiA, and ubiG involved in UQ biosynthesis in E. coli, to construct a better co-expression system. The expression coupled by dps and ubiCA was effective for increasing UQ-10 production by five times than that by expressing single dps gene in the shake flask culture. To study for a large-scale production of UQ-10 in E. coli, fed-batch fermentations were implemented to achieve a high cell density culture. A cell concentration of 85.40 g/L and 94.58 g/L dry cell weight (DCW), and UQ-10 content of 50.29 mg/L and 45.86 mg/L was obtained after 32.5 h and 27.5 h of cultivation, subsequent to isopropyl-β-d-thiogalactopy ranoside and lactose induction, respectively. In addition, plasmid stability was maintained at high level throughout the fermentation.     

Effect of endogenous ubiquinone on the interaction of exogenous Ubiquinone-1 with the respiratory chain of bovine heart mitochondria

Pentane extraction of lyophilized mitochondria with depletion of up to 92% of endogenous ubiquinone (UQ) does not affect ubiquinol oxidase activity in terms of K_m; in certain preparations the V is decreased probably because the extraction is harmful to the membrane integrity. In such case dl-α-tocopherol is able to maintain enzymatic activity up to the normal values found in control mitochondria. On the other hand, NADH-UQ-1 reductase activity is greatly affected by pentane extraction with a large decrease in V but no change in K_m, but this activity is protected by addition of dl-α-tocopherol to the extraction medium. The same conclusions can be drawn for succinate-UQ-1 reductase activity. In conclusion it appears that endogenous UQ does not mediate the interaction of exogenous UQ-1 with the redox sites for UQ in the respiratory chain.     

Kinetics and Mechanism of the Reaction of a Nitroxide Radical (Tempol) With a Phenolic Antioxidant

In the absence of redox-active transition metal ions, the removal of Tempol by Trolox occurs by a simple bimolecular reaction that, most probably, involves a hydrogen transfer from phenol to nitroxide. The specific rate constant of the process is small (0.1 M &#109 1 s &#109 1 ). Metals can catalyze the process, as evidenced by the decrease in rate observed in the presence of diethylenetriaminepentaacetic acid (DTPA). Furthermore, addition of Fe(II) (20 &#119 M ferrous sulfate and 40 &#119 M EDTA) produces a noticeable increase in the rate of Tempol consumption.     

Regulation of MUC5AC expression by NAD(P)H:quinone oxidoreductase 1

Neutrophil elastase (NE), a potent neutrophil inflammatory mediator, increases MUC5AC mucin gene expression through undefined pathways involving reactive oxygen species. To determine the source of NE-generated reactive oxygen species, we used pharmacologic inhibitors of oxidoreductases to test whether they blocked NE-regulated MUC5AC mRNA expression. We found that dicumarol, an inhibitor of the NADP(H):quinone oxidoreductase 1 (NQO1), inhibited MUC5AC mRNA expression in A549 lung adenocarcinoma cells and primary normal human bronchial epithelial cells. We further tested the role of NQO1 in mediating NE-induced MUC5AC expression by inhibiting NQO1 expression using short interfering RNA (siRNA). Transfection with siRNA specific for NQO1 suppressed NQO1 expression and significantly abrogated MUC5AC mRNA expression. NE treatment caused lipid peroxidation in A549 cells; this effect was inhibited by pretreatment with dicumarol, suggesting that NQO1 also regulates oxidant stress in A549 cells after NE exposure. NE exposure increased NQO1 protein and activity levels; NQO1 expression and activity were limited to the cytosol and did not translocate to the plasma membrane. Our results indicate that NQO1 has an important role as a key mediator of NE-regulated oxidant stress and MUC5AC mucin gene expression.     

Genetic evidence for NAD(P)H:quinone oxidoreductase 1-catalyzed quinone reduction on passage through the mouse pulmonary circulation

The quinones duroquinone (DQ) and coenzyme Q(1) (CoQ(1)) and quinone reductase inhibitors have been used to identify reductases involved in quinone reduction on passage through the pulmonary circulation. In perfused rat lung, NAD(P)H:quinone oxidoreductase 1 (NQO1) was identified as the predominant DQ reductase and NQO1 and mitochondrial complex I as the CoQ(1) reductases. Since inhibitors have nonspecific effects, the goal was to use Nqo1-null (NQO1(-)/(-)) mice to evaluate DQ as an NQO1 probe in the lung. Lung homogenate cytosol NQO1 activities were 97 ± 11, 54 ± 6, and 5 ± 1 (SE) nmol dichlorophenolindophenol reduced·min(-1)·mg protein(-1) for NQO1(+/+), NQO1(+/-), and NQO1(-/-) lungs, respectively. Intact lung quinone reduction was evaluated by infusion of DQ (50 μM) or CoQ(1) (60 μM) into the pulmonary arterial inflow of the isolated perfused lung and measurement of pulmonary venous effluent hydroquinone (DQH(2) or CoQ(1)H(2)). DQH(2) efflux rates for NQO1(+/+), NQO1(+/-), and NQO1(-/-) lungs were 0.65 ± 0.08, 0.45 ± 0.04, and 0.13 ± 0.05 (SE) μmol·min(-1)·g dry lung(-1), respectively. DQ reduction in NQO1(+/+) lungs was inhibited by 90 ± 4% with dicumarol; there was no inhibition in NQO1(-/-) lungs. There was no significant difference in CoQ(1)H(2) efflux rates for NQO1(+/+) and NQO1(-/-) lungs. Differences in DQ reduction were not due to differences in lung dry weights, wet-to-dry weight ratios, perfusion pressures, perfused surface areas, or total DQ recoveries. The data provide genetic evidence implicating DQ as a specific NQO1 probe in the perfused rodent lung.     

Current prospects for the production of coenzyme Q10 in microbes

Coenzyme Q or ubiquinone (UQ) is a naturally occurring coenzyme formed from the conjugation of a benzoquinone ring and an isoprenoid chain of varying length. UQ-10, the main UQ species produced by humans, provides therapeutic benefits in certain human diseases, such as cardiomyopathy, when administered orally. Increased consumer demand has led to the development of bioprocesses for the commercial production of UQ-10. Up to now, these processes have relied on microbes that produce high levels of UQ-10 naturally. However, as knowledge of the biosynthetic enzymes and of regulatory mechanisms modulating UQ production increases, opportunities arise for the genetic engineering of UQ-10 production in hosts, such as Escherichia coli, that are better suited for commercial fermentation. We present the various strategies used up to now to improve and/or engineer UQ-10 production in microbes and analyze yields obtained in light of the current knowledge on the biosynthesis of this molecule.     

The kinetics of NADH oxidation by complex I of isolated plant mitochondria

The oxidation of matrix NADH in the presence and absence of rotenone was investigated in submitochondrial particles prepared from purified beetroot ( Beta vulgaris L.) mitochondria. The submitochondrial particles oxidised NADH using oxygen and artificial electron acceptors such as ferricyanide (FeCN) and short-chain analogues of ubiquinone(UQ)-10, although the NADH-FeCN reductase activity was not inhibited by rotenone. NADH-oxygen reductase activity in the presence and absence of rotenone displayed different affinities for NADH (145 ± 37 and 24 ± 9 μ M , respectively). However, in the presence of 0.15 m M UQ-1 where any contribution from non-specific sites of UQ-reduction was minimal, the rotenone-insensitive oxygen uptake was stimulated dramatically and the K_m(NADH) decreased from 167 ± 55 μ M to 11 ± 1 μ M ; a value close to that determined for the total oxygen uptake which itself was virtually unaffected by the addition of UO-1 [K_m(NADH) of 13 ± 3 μ M ).
The similar affinity of NADH-oxygen reductase for NADH when UQ-1 was present in both the presence and absence of rotenone, suggested that there may be only one NADH binding site involved in the two activities. A quantitative two-stage model for Complex I is postulated with one NADH binding site and two sites of UQ-reduction (one of which is insensitive to rotenone) with a common intermediate 'P' whose level of reduction can influence the NADH binding site. The poor affinity that rotenone-insensitive NADH-oxygen reductase activity displayed for NADH results from a limitation on the interaction of its UQ-reduction site with UQ-10 in the membrane; possibly from a low concentration of UQ-10 around this site or from steric hindrance restricting the access of UQ-10 to this reduction site.     

PGE 1 Protection against Apoptosis Induced by d -galactosamine is Not Related to the Modulation of Intracellular Free Radical Production in Primary Culture of Rat Hepatocytes

d -galactosamine ( d -GalN) toxicity is a useful experimental model of liver failure in human. It has been previously observed that PGE 1 treatment reduced necrosis and apoptosis induced by d -GalN in rats. Primary cultured rat hepatocytes were used to evaluate if intracellular oxidative stress was involved during the induction of apoptosis and necrosis by d -GalN (0-40 mM). Also, the present study investigated if PGE 1 (1 &#119 M) was equally potent reducing both types of cell death. The presence of hypodiploid cells, DNA fragmentation and caspase-3 activation were used as a marker of hepatocyte apoptosis. Necrosis was measured by lactate dehydrogenase (LDH) release. Oxidative stress was evaluated by the intracellular production of hydrogen peroxide (H 2 O 2 ), the disturbances on the mitochondrial transmembrane potential (MTP), thiobarbituric-reacting substances (TBARS) release and the GSH/GSSG ratio. Data showed that intermediate range of d -GalN concentrations (2.5-10 mM) induced apoptosis in association with a moderate oxidative stress. High d -GalN concentration (40 mM) induced a reduction of all parameters associated with apoptosis and enhanced all those related to necrosis and intracellular oxidative stress, including a reduction of GSH/GSSG ratio and MTP in comparison with d -GalN (2.5-10 mM)-treated cells. Although PGE 1 reduced apoptosis induced by d -GalN, it was not able to reduce the oxidative stress and cell necrosis induced by the hepatotoxin in spite to its ability to abolish the GSH depletion.     

Inhibition of ubiquinone synthesis in isolated rat heart under an ischemic condition

1. The biosynthesis of ubiquinone (UQ) in isolated rat heart under ischemic and hypoxic conditions was investigated. 2. Under ischemic perfusion, a greater amount of biosynthetic intermediates, 3-nonaprenyl and 3-decaprenyl-4-hydroxybenzoate (PPHBs) was accumulated and a smaller amount of UQ-9 and -10 was synthesized when compared with normal conditions. 3. The accumulation of PPHBs was observed without forming UQs during anaerobic perfusion. 4. Hydroxylation which is the following reaction of PPHBs for the biosynthesis of UQ in rat heart, was proceeded by the monooxygenase(s) depending upon the oxygen concentrations.     

Antioxidant α-Keto-carboxylate Pyruvate Protects Low-density Lipoprotein and Atherogenic Macrophages

Oxidative modification of low-density lipoprotein (oxLDL) plays a pathogenic role in atherogenesis. Classical antioxidants such as l -ascorbic acid can inhibit formation of oxLDL. &#102 -Keto-carboxylates such as pyruvate and congeners also display antioxidant properties in some cell-free and intact cell systems. We tested the hypothesis that pyruvate or &#102 -keto-glutarate may function as antioxidants with respect to LDL incubated with 5 or 10 &#119 M Cu 2+ alone or in combination with THP-1-derived macrophages. &#102 -Hydroxy-carboxylates ( l -lactate), linear aliphatic mono-carboxylates (acetate/caprylate) and l -ascorbic acid served as controls. The oxLDL formation was ascertained by electrophoretic mobility and oxLDL cytotoxicity was judged by macrophage viability and thiobarbituric acid reactive substances (TBARS) formation. Cu 2+ alone was not cytotoxic but increased electrophoretic mobility of cell-free LDL, stimulating TBARS. Millimolar pyruvate, &#102 -keto-glutarate, or micromolar l -ascorbic acid partially inhibited oxLDL formation, while &#102 -hydroxy-carboxylate or the aliphatic mono-carboxylates had no measurable antioxidant properties in cell-free LDL. Co-culture of LDL with macrophages and Cu 2+ augmented TBARS release and resulted in 95% macrophage death. Pyruvate improved macrophage viability with 5 &#119 M Cu 2+ up to 60%. l -Ascorbic acid ( &#83 100 &#119 M) protected macrophages up to 80%. When &#83 100 &#119 M l -ascorbic acid was combined with pyruvate, oxLDL formation and macrophage death were fully prevented. Thus, &#102 -keto-carboxylates, but not physiological &#102 -hydroxy-carboxylates or aliphatic mono-carboxylates qualify as antioxidants in LDL systems. Since &#102 -keto-carboxylates enhanced the antioxidant power of l -ascorbic acid, our findings may have implications for strategies attenuating atherosclerosis.     

Engineering of ubiquinone biosynthesis using the yeast coq2 gene confers oxidative stress tolerance in transgenic tobacco

Ubiquinone (UQ), an electron carrier in the respiratory chain ranging from bacteria to humans, shows antioxidative activity in vitro, but its physiological role in vivo is not yet clarified in plants. UQ biosynthesis was modified by overexpressing the yeast gene coq2, which encodes p-hydroxybenzoate:polyprenyltransferase, to increase the accumulation of UQ-6 in yeast and UQ-10 in tobacco. The yeast and tobacco transgenic lines showed about a three- and six-fold increase in UQ, respectively. COQ2 polypeptide, the localization of which was forcibly altered to the endoplasmic reticulum, had the same or a greater effect as mitochondria-localized COQ2 on the increase in UQ in both the yeast and tobacco transformants, indicating that the UQ intermediate is transported from the endoplasmic reticulum to the mitochondria. Plants with a high UQ level are more resistant to oxidative stresses caused by methyl viologen or high salinity. This is attributable to the greater radical scavenging ability of the transgenic lines when compared with the wild type.     

Cyanidin-3- O -β-glucopyranoside Protects Myocardium and Erythrocytes from Oxygen Radical-mediated Damages

The cyanidin-3- O - &#103 -glucopyranoside (C-3-G) antioxidant capacity towards reactive oxygen species (ROS)-mediated damages was assessed in tissue and cells submitted to increased oxidative stress. In the isolated ischemic and reperfused rat heart, 10 or 30 &#119 M C-3-G protected from both lipid peroxidation (66.7 and 94% inhibition of malondialdehyde (MDA) generation in 10 and 30 &#119 M C-3-G-reperfused hearts, respectively, in comparison with control reperfused hearts) and energy metabolism impairment (higher ATP concentration in 10 and 30 &#119 M C-3-G-reperfused hearts than in control reperfused hearts). These effects were associated to C-3-G permeation within myocardial cells, as indicated by results obtained in the isolated rat heart perfused for 30 min in the recirculating Langendorff mode under normoxia with 10 and 30 &#119 M C-3-G. Protective effects were exerted, in a dose-dependent manner, by C-3-G also in 2 mM hydrogen peroxide-treated human erythrocytes. With respect to MDA formation, an apparent IC 50 of 5.12 &#119 M was calculated for C-3-G (the polyphenol resveratrol used for comparison showed an apparent IC 50 of 38.43 &#119 M). The general indications are that C-3-G (largely diffused in dietary plants and fruits, such as pigmented oranges very common in the Mediterranean diet) represents a powerful natural antioxidant with beneficial effects in case of increased oxidative stress, and at pharmacological concentrations it is able to decrease tissue damages occurring in myocardial ischemia and reperfusion.     

Expression of NAD(P)H:quinone oxidoreductase 1 in HeLa cells: role of hydrogen peroxide and growth phase

The aim of this work was to study the role of H(2)O(2) in the regulation of NAD(P)H:quinone oxidoreductase 1 (NQO1, DT-diaphorase, EC ) with relation to cell density of HeLa cells cultures and the function played by NQO1 in these cells. Levels of NQO1 activity were much higher (40-fold) in confluent HeLa cells than in sparse cells, the former cells being much more resistant to H(2)O(2). Addition of sublethal concentrations of H(2)O(2) (up to 24 microm) produced a significant increase of NQO1 (up to 16-fold at 12 microm) in sparse cells but had no effect in confluent cells. When cells reached confluency in the presence of pyruvate, a H(2)O(2) scavenger, NQO1 activity was decreased compared with cultures grown to confluency without pyruvate. Inhibition of quinone reductases by dicumarol substantially decreased viability of confluent cells in serum-free medium. This is the first demonstration that regulation of NQO1 expression by H(2)O(2) is dependent on the cell density in HeLa cells and that endogenous generation of H(2)O(2) participates in the increase of NQO1 activity as cell density is higher. This enzyme is required to promote survival of confluent cells.     

Role of NAD(P)H:quinone oxidoreductase (NQO1) in apoptosis induction by aziridinylbenzoquinones RH1 and MeDZQ

We aimed to characterize the role of NAD(P)H:quinone oxidoreductase (NQO1) in apoptosis induction by antitumour quinones RH1 (2,5-diaziridinyl-3-hydroxymethyl-6-methyl-1,4-benzoquinone) and MeDZQ (2,5-dimethyl-3,6-diaziridinyl-1,4-benzoquinone). Digitonin-permeabilized FLK cells catalyzed NADPH-dependent single- and two-electron reduction of RH1 and MeDZQ. At equitoxic concentrations, RH1 and MeDZQ induced apoptosis more efficiently than the nonalkylating duroquinone or H(2)O(2). The antioxidant N,N'-diphenyl-p-phenylene diamine, desferrioxamine, and the inhibitor of NQO1 dicumarol, protected against apoptosis induction by all compounds investigated, but to a different extent. The results of multiparameter regression analysis indicate that RH1 and MeDZQ most likely induce apoptosis via NQO1-linked formation of alkylating species but not via NQO1-linked redox cycling.