Cytotoxicity of RH1 and related aziridinylbenzoquinones: involvement of activation by NAD(P)H:quinone oxidoreductase (NQO1) and oxidative stress

It is supposed that the main cytotoxicity mechanism of antitumour aziridinyl-substituted benzoquinones is their two-electron reduction to alkylating products by NAD(P)H:quinone oxidoreductase (NQO1, DT-diaphorase, EC 1.6.99.2). However, other possible cytotoxicity mechanisms, e.g., oxidative stress, are studied insufficiently. In the single-electron reduction of quinones including a novel compound RH1 (2,5-diaziridinyl- 3-(hydroxymethyl)-6-methyl-1,4-benzoquinone), by NADPH:cytochrome P-450 reductase (EC 1.6.2.4, P-450R), their reactivity increased with an increase in the redox potential of quinone/semiquinone couple (E(1)7), reaching a limiting value at E(1)7> or =-0.1V. The reactivity of quinones towards NQO1 did not depend on their E(1)7. The cytotoxicity of aziridinyl-unsubstituted quinones in bovine leukemia virus-transformed lamb kidney fibroblasts (line FLK) mimics their reactivity in P-450R-catalyzed reactions, exhibiting a parabolic dependence on their E(1)7. The toxicity of aziridinyl-benzoquinones, although being higher, also followed this trend and did not depend on their reactivity towards NQO1. The action of aziridinylbenzoquinones in FLK cells was accompanied by an increase in lipid peroxidation, their toxicity decreased by desferrioxamine and the antioxidant N,N'-diphenyl-p-phenylene diamine, and potentiated by 1,3-bis-(2-chloroethyl)-1-nitrosourea. The inhibitor of NQO1, dicumarol, protected against the toxicity of aziridinyl-benzoquinones except of 2,5-bis-(2'-hydroxyethylamino)-3,6-diaziridinyl-1,4-benzoquinone (BZQ), which was almost inactive as NQO1 substrate. The same events except the absence of pronounced effect of dicumarol were characteristic in the cytotoxicity of aziridinyl-unsubstituted quinones. These findings indicate that in addition to the activation by NQO1, the oxidative stress presumably initiated by single-electron transferring enzymes may be an important factor in the cytotoxicity of aziridinylbenzoquinones. The information obtained may contribute to the understanding of the molecular mechanisms of aziridinylquinone cytotoxicity and may be useful in the design of future bioreductive drugs.     

Immunodetection of NAD(P)H:quinone oxidoreductase 1 (NQO1) in human tissues

Despite the extensive interest in NADPH:quinone oxidoreductase (NQO1, DT-diaphorase), there is little immunohistochemical information regarding its distribution in either normal human tissues or in human tumors. Using immunohistochemistry (IHC), we have examined cell-specific expression of NQO1 in many normal tissues and tumors as a step toward defining the distribution of NQO1 in humans. NQO1 was detected by IHC in respiratory, breast duct, thyroid follicle, and colonic epithelium, as well as in the corneal and lens epithelium of the eye. NQO1 was also detected by IHC in vascular endothelium in all tissues examined. NQO1 could also readily be detected in the endothelial lining of the aorta but was not detected using immunoblot analysis in the myocardium. Adipocytes stained positive for NQO1, and the enzyme was also detected by both IHC and immunoblot analysis in parasympathetic ganglia in the small intestine and in the optic nerve and nerve fibers. NQO1 was not highly expressed in five different human liver samples using immunoblot analysis, whereas studies using IHC demonstrated only trace NQO1 staining in isolated bile duct epithelium. NQO1 expresion was also examined by IHC in a variety of solid tumors. Marked NQO1 staining was detected in solid tumors from thyroid, adrenal, breast, ovarian, colon, and cornea and in non-small cell lung cancers. The NQO1 content of many solid tumors supports the use of NQO1-directed anticancer agents for therapeutic purposes, but the distribution of NQO1 in normal tissues suggests that potential adverse effects of such agents need to be carefully monitored in preclinical studies.     

Novel quinolinequinone antitumor agents: structure-metabolism studies with NAD(P)H:quinone oxidoreductase (NQO1)

A series of quinolinequinones bearing various substituents has been synthesized, and the effects of substituents on the metabolism of the quinones by recombinant human NAD(P)H:quinone oxidoreductase (hNQO1) was studied. A range of quinolinequinones were selected for study, and were specifically designed to probe the effects of aryl substituents at C-2. A range of 28 quinolinequinones 2-29 was prepared using three general strategies: the palladium(0) catalyzed coupling of 2-chloroquinolines, the classical Friedl?nder synthesis and the double-Vilsmeier reaction of acetanilides. One example of an isoquinolinequinone 30 was also prepared, and the reduction potentials of the quinones were measured by cyclic voltammetry. For simple substituents R(2) at the quinoline 2-position, the rates of quinone metabolism by hNQO1 decrease for R(2)=Cl>H approximately Me>Ph. For aromatic substituents, the rate of reduction decreases dramatically for R(2)=Ph>1-naphthyl>2-naphthyl>4-biphenyl. Compounds containing a pyridine substituent are the best substrates, and the rates decrease as R(2)=4-pyridyl>3-pyridyl>2-pyridyl>4-methyl-2-pyridyl>5-methyl-2-pyridyl. The toxicity toward human colon carcinoma cells with either no detectable activity (H596 or BE-WT) or high NQO1 activity (H460 or BE-NQ) was also studied in representative quinones. Quinones that are good substrates for hNQO1 are more toxic to the NQO1 containing or expressing cell lines (H460 and BE-NQ) than the NQO1 deficient cell lines (H596 and BE-WT).     

Novel quinolinequinone antitumor agents: structure-metabolism studies with NAD(P)H:quinone oxidoreductase (NQO1).

The effects of functional group changes on the metabolism of novel quinolinequinones by recombinant human NAD(P)H:quinone oxidoreductase (NQO1) are described. Overall, the quinolinequinones were much better substrates for NQO1 than analogous indolequinones, with compounds containing heterocyclic substituents at C-2 being among the best substrates.     

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.     

NAD(P)H:quinone acceptor oxidoreductase 1 (NQO1), a multifunctional antioxidant enzyme and exceptionally versatile cytoprotector

NAD(P)H:quinone acceptor oxidoreductase 1 (NQO1) is a widely-distributed FAD-dependent flavoprotein that promotes obligatory 2-electron reductions of quinones, quinoneimines, nitroaromatics, and azo dyes, at rates that are comparable with NADH or NADPH. These reductions depress quinone levels and thereby minimize opportunities for generation of reactive oxygen intermediates by redox cycling, and for depletion of intracellular thiol pools. NQO1 is a highly-inducible enzyme that is regulated by the Keap1/Nrf2/ARE pathway. Evidence for the importance of the antioxidant functions of NQO1 in combating oxidative stress is provided by demonstrations that induction of NQO1 levels or their depletion (knockout, or knockdown) are associated with decreased and increased susceptibilities to oxidative stress, respectively. Furthermore, benzene genotoxicity is markedly enhanced when NQO1 activity is compromised. Not surprisingly, human polymorphisms that suppress NQO1 activities are associated with increased predisposition to disease. Recent studies have uncovered protective roles for NQO1 that apparently are unrelated to its enzymatic activities. NQO1 binds to and thereby stabilizes the important tumor suppressor p53 against proteasomal degradation. Indeed, NQO1 appears to regulate the degradative fate of other proteins. These findings suggest that NQO1 may exercise a selective “gatekeeping” role in regulating the proteasomal degradation of specific proteins, thereby broadening the cytoprotective role of NQO1 far beyond its highly effective antioxidant functions.     

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 (CoQ1) 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 CoQ1 cytoprotective mechanism required CoQ1 reduction by DT-diaphorase (NQO1). Furthermore, the mitochondrial membrane potential and ATP levels were restored at low CoQ1 concentrations (5 microM). This suggests that the CoQ1H2 formed by NQO1 reduced complex III and acted as an electron bypass of the rotenone block. However cytoprotection still occurred at higher CoQ1 concentrations (>10 microM), 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 CoQ1 or menadione cytoprotection also involves the NQO1 catalysed reoxidation of NADH that accumulates as a result of complex I inhibition. The CoQ1H2 formed would then also act as a ROS scavenger.     

NAD(P)H: quinone oxidoreductase 1 expression in kidney podocytes.

NAD(P)H:quinone oxidoreductase 1 (NQO1; DT-diaphorase; DTD) is a cytosolic two-electron reductase, and compounds of the quinone family such as mitomycin C are efficiently bioactivated by this enzyme. The observation that DT-diaphorase is highly expressed in many cancerous tissues compared to normal tissues has provided us with a potentially selective target that can be exploited in the design of novel anticancer agents. Because of the relative lack of information about the cell-specific expression of DT-diaphorase, the purpose of this study was to map the distribution of this enzyme in normal human tissues. Fifteen tissue samples from normal human kidney were analyzed for expression of DT-diaphorase by immunohistochemistry (two-step indirect method). We found a specific high expression of DT-diaphorase in glomerular visceral epithelial cells (podocytes). These results suggest that a high expression of DT-diaphorase in podocytes could play a major role in the pathogenesis of renal toxicity and mitomycin C-induced hemolytic uremic syndrome, in which injury to the glomerular filtration mechanism is the primary damage, leading to a cascade of deleterious events including microangiopathic hemolytic anemia and thrombocytopenia. This observation has potential therapeutic implications because the DT-diaphorase metabolic pathway is influenced by many agents, including drugs, diet, and environmental cell factors such as pH and oxygen tension.     

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.     

L-Dopa stimulates expression of the antioxidant enzyme NAD(P)H:quinone oxidoreductase (NQO) in cultured astroglial cells

The autooxidation of L-Dopa, a catecholamine used in the symptomatic treatment of Parkinson's disease, generally yields reactive oxygen species and neurotoxic quinones. NAD(P)H:quinone oxidoreductase (NQO) is a flavoenzyme that is implicated in the detoxication of quinones, including those formed during L-Dopa autooxidation. Through the action of this enzyme, deleterious redox-labile quinones are turned into less toxic and more stable hydroquinones that are amenable to further detoxication and/or cellular excretion. In the present study, using primary rat astrocytes and C6 astroglioma as a model to evaluate the neuroprotective response of astroglial cells upon exposure to L-Dopa, we demonstrate that this compound, or more correctly its quinone (auto)oxidation products, up-regulates astroglial NQO in a time- and concentration-dependent way as assessed at the level of mRNA expression, protein level, and enzymatic activity. Moreover, under similar conditions cellular glutathione content was enhanced. It is concluded that, similar to glutathione, the oxidative stress limiting NQO is likely to contribute to the capacity of astroglial cells to protect dopaminergic neurons against L-Dopa, and, hence, may be considered as a potential target for the development of neuroprotective strategies for Parkinson's disease.     

Human NAD(P)H:quinone oxidoreductase inhibition by flavonoids in living cells

Procedures for assessing enzyme inhibition in living cells are an important tool in the study of the relevance of enzyme-catalyzed reactions and interactions in the human body. This paper presents the effects of flavonoids on NAD(P)H:quinone oxidoreductase 1 (NQO1) activity, by a newly developed method to measure NQO1 inhibition in intact cells. The principle of this method is based on the resorufin reductase activity of NQO1. The change in fluorescence in time was used to determine NQO1 activity in intact Chinese hamster ovary (CHO) cells genetically engineered to overexpress human NQO1. Applying this method to determine the inhibitory effects of reported in vitro NQO1 inhibitors (dicoumarol, 7,8-dihydroxyflavone, chrysin) showed that for all inhibitors tested, the IC50 in intact cells was at least 3 orders of magnitude higher than the IC50 in cell lysates. This result demonstrates that in vitro studies with purified NQO1 or with extracts from disrupted tissues are of limited value for obtaining insight into the situation in living cells. Possible factors underlying this discrepancy are being discussed. For the first time, we determined NQO1 inhibition by flavonoids in cells without disruption of the cells or addition of cofactors, enabling the assessment of enzymatic activity and the interaction of modulators of enzymatic activity in an intracellular situation.     

Overexpression of NAD(P)H:quinone oxidoreductase 1 in human reproductive system.

NAD(P)H:quinone oxidoreductase 1 (NQO1; DT-diaphorase; DTD) is a two-electron reductase that efficiently bioactivates compounds of the quinone family, such as mitomycin C. The observation that DTD is overexpressed in many cancerous tissues compared to normal tissues has provided us with a potentially selective target that can be exploited in the design of novel anticancer agents. Because of the relative lack of information on the cell-specific expression of DTD, the purpose of this study was to perform a body mapping of its normal distribution. Tissue samples from various components of the human reproductive system were analyzed by immunohistochemistry. We found strong expression of this enzyme in testicular stromal cells (Leydig cells) and in the epithelium of epididymis, ductuli efferentes, and Fallopian tube. These results suggest that DTD-bioactivated quinones could be responsible for a selective toxicity on these components of the reproductive system and cause clinical problems due to testosterone deficiency and infertility. This observation needs to be investigated in preclinical evaluation of new anticancer quinones and in patients treated with these compounds. (J Histochem Cytochem 49:1187-1188, 2001)     

NAD(P)H:quinone oxidoreductase 1 protects lungs from oxidant-induced emphysema in mice

Emphysema is currently a leading cause of mortality with no known effective therapy to attenuate progressive loss of lung function. Previous work supports that activation of nuclear factor erythroid 2-related factor 2 (Nrf2) is protective to the lung through induction of hundreds of antioxidant genes. In models of lung injury, the expression of NAD(P)H:quinone oxidoreductase 1 (NQO1) is upregulated in a manner dependent on Nrf2 and human emphysema is associated with reduced levels of NQO1. However, the functional role of NQO1 in emphysema remains unknown. In this study, we demonstrate the protective role of NQO1 in the development of emphysema using mouse models. NQO1-deficient animals demonstrated premature age-related emphysema and were more susceptible to both elastase and inhaled lipopolysaccharide models of emphysema. The absence of NQO1 was associated with enhanced markers of oxidant stress. Treatment of NQO1-deficient animals with the antioxidant N-acetylcysteine reversed the NQO1-dependent emphysematous changes. In vitro studies utilizing either inhibition or induction of NQO1 demonstrated a potent antioxidant role of NQO1 in macrophages, suggesting a role for macrophage-derived oxidants in the pathogenesis of emphysema. These novel findings support a functional role for NQO1 in protecting the lung from development of emphysema.     

NAD(P)H quinone oxidoreductase 1 regulates neutrophil elastase-induced mucous cell metaplasia

Mucous cell metaplasia (MCM) and neutrophil-predominant airway inflammation are pathological features of chronic inflammatory airway diseases. A signature feature of MCM is increased expression of a major respiratory tract mucin, MUC5AC. Neutrophil elastase (NE) upregulates MUC5AC in primary airway epithelial cells by generating reactive oxygen species, and this response is due in part to upregulation of NADPH quinone oxidoreductase 1 (NQO1) activity. Delivery of NE directly to the airway triggers inflammation and MCM and increases synthesis and secretion of MUC5AC protein from airway epithelial cells. We hypothesized that NE-induced MCM is mediated in vivo by NQO1. Male wild-type and Nqo1-null mice (C57BL/6 background) were exposed to human NE (50 μg) or vehicle via oropharyngeal aspiration on days 1, 4, and 7. On days 8 and 11, lung tissues and bronchoalveolar lavage (BAL) samples were obtained and evaluated for MCM, inflammation, and oxidative stress. MCM, inflammation, and production of specific cytokines, granulocyte-macrophage colony-stimulating factor, macrophage inflammatory protein-2, interleukin-4, and interleukin-5 were diminished in NE-treated Nqo1-null mice compared with NE-treated wild-type mice. However, in contrast to the role of NQO1 in vitro, we demonstrate that NE-treated Nqo1-null mice had greater levels of BAL and lung tissue lipid carbonyls and greater BAL iron on day 11, all consistent with increased oxidative stress. NQO1 is required for NE-induced inflammation and MCM. This model system demonstrates that NE-induced MCM directly correlates with inflammation, but not with oxidative stress.     

Evidence for NAD(P)H:quinone oxidoreductase 1 (NQO1)-mediated quinone-dependent redox cycling via plasma membrane electron transport: A sensitive cellular assay for NQO1

2,3-Dimethoxy 1,4-naphthoquinone (DMNQ), which redox cycles via two-electron reduction, mediates reduction of the cell-impermeative tetrazolium dye WST-1 in kidney epithelial cells (MDCK), which express high levels of NQO1, but not in HL60 or CHO cells, which are NQO1 deficient. DMNQ-dependent WST-1 reduction by MDCK cells was strongly inhibited by low concentrations of the NQO1 inhibitor dicoumarol and was also inhibited by diphenyleneiodonium, capsaicin, and superoxide dismutase (SOD), but not by the uncoupler FCCP or the complex IV inhibitor cyanide. This suggests that DMNQ-dependent WST-1 reduction by MDCK cells is catalyzed by NQO1 via redox cycling and plasma membrane electron transport (PMET). Interestingly, we observed an association between DMNQ/WST-1 reduction and extracellular H₂O₂ production as determined by Amplex red. Exposure of MDCK cells to DMNQ for 48 h caused cellular toxicity that was extensively reversed by co-incubation with dicoumarol or exogenous SOD, catalase, or N-acetylcysteine. No effects were observed in NQO1-deficient CHO and HL60 cells. In conclusion, we have developed a simple real-time cellular assay for NQO1 and show that PMET plays a significant role in DMNQ redox cycling via NQO1, leading to cellular toxicity in cells with high NQO1 levels.