NAD(P)H Quinone Dehydrogenase 1 Ablation Inhibits Activation of the Phosphoinositide 3-Kinase/Akt Serine/Threonine Kinase and Mitogen-Activated Protein Kinase/Extracellular Signal-Regulated Kinase Pathways and Blocks Metabolic Adaptation in Hepatocellular Carcinoma.

Cancer cells undergo metabolic adaptation to sustain uncontrolled proliferation. Aerobic glycolysis and glutaminolysis are two of the most essential characteristics of cancer metabolic reprogramming. Hyperactivated phosphoinositide 3-kinase (PI3K)/Akt serine/threonine kinase (Akt) and mitogen-activated protein kinase (MAPK)/extracellular signal-regulated kinase (ERK) signaling pathways play central roles in cancer cell metabolic adaptation given that their downstream effectors, such as Akt and c-Myc, control most of the glycolytic and glutaminolysis genes. Here, we report that the cytosolic flavoprotein, NAD(P)H quinone dehydrogenase 1 (Nqo1), is strongly overexpressed in mouse and human hepatocellular carcinoma (HCC). Knockdown of Nqo1 enhanced activity of the serine/threonine phosphatase, protein phosphatase 2A, which operates at the intersection of the PI3K/Akt and MAPK/ERK pathways and dephosphorylates and inactivates pyruvate dehydrogenase kinase 1, Akt, Raf, mitogen-activated protein kinase kinase, and ERK1/2. Nqo1 ablation also induced the expression of phosphatase and tensin homolog, a dual protein/lipid phosphatase that blocks PI3K/Akt signaling, through the ERK/cAMP-responsive element-binding protein/c-Jun pathway. Together, Nqo1 ablation triggered simultaneous inhibition of the PI3K/Akt and MAPK/ERK pathways, suppressed the expression of glycolysis and glutaminolysis genes and blocked metabolic adaptation in liver cancer cells. Conversely, Nqo1 overexpression caused hyperactivation of the PI3K/Akt and MAPK/ERK pathways and promoted metabolic adaptation. Conclusion: In conclusion, Nqo1 functions as an upstream activator of both the PI3K/Akt and MAPK/ERK pathways in liver cancer cells, and Nqo1 ablation blocked metabolic adaptation and inhibited liver cancer cell proliferation and HCC growth in mice. Therefore, our results suggest that Nqo1 may function as a therapeutic target to inhibit liver cancer cell proliferation and inhibit HCC.

Reduction of 2-methoxy-1,4-naphtoquinone by mitochondrially-localized Nqo1 yielding NAD+ supports substrate-level phosphorylation during respiratory inhibition.

Provision of NAD+ for oxidative decarboxylation of alpha-ketoglutarate to succinyl-CoA by the ketoglutarate dehydrogenase complex (KGDHC) is critical for maintained operation of succinyl-CoA ligase yielding high-energy phosphates, a process known as mitochondrial substrate-level phosphorylation (mSLP). We have shown previously that when NADH oxidation by complex I is inhibited by rotenone or anoxia, mitochondrial diaphorases yield NAD+, provided that suitable quinones are present (Kiss G et al., FASEB J 2014, 28:1682). This allows for KGDHC reaction to proceed and as an extension of this, mSLP. NAD(P)H quinone oxidoreductase 1 (NQO1) is an enzyme exhibiting diaphorase activity. Here, by using Nqo1-/- and WT littermate mice we show that in rotenone-treated, isolated liver mitochondria 2-methoxy-1,4-naphtoquinone (MNQ) is preferentially reduced by matrix Nqo1 yielding NAD+ to KGDHC, supporting mSLP. This process was sensitive to inhibition by specific diaphorase inhibitors. Reduction of idebenone and its analogues MRQ-20 and MRQ-56, menadione, mitoquinone and duroquinone were unaffected by genetic disruption of the Nqo1 gene. The results allow for the conclusions that i) MNQ is a Nqo1-preferred substrate, and ii) in the presence of suitable quinones, mitochondrially-localized diaphorases other than Nqo1 support NADH oxidation when complex I is inhibited. Our work confirms that complex I bypass can occur by quinones reduced by intramitochondrial diaphorases oxidizing NADH, ultimately supporting mSLP. Finally, it may help to elucidate structure-activity relationships of redox-active quinones with diaphorase enzymes.

Role of the NAD(P)H quinone oxidoreductase NQR and the cytochrome b AIR12 in controlling superoxide generation at the plasma membrane.

MAIN CONCLUSION: The quinone reductase NQR and the b-type cytochrome AIR12 of the plasma membrane are important for the control of reactive oxygen species in the apoplast. AIR12 and NQR are two proteins attached to the plant plasma membrane which may be important for generating and controlling levels of reactive oxygen species in the apoplast. AIR12 (Auxin Induced in Root culture) is a single gene of Arabidopsis that codes for a mono-heme cytochrome b. The NADPH quinone oxidoreductase NQR is a two-electron-transferring flavoenzyme that contributes to the generation of O 2•- in isolated plasma membranes. A. thaliana double knockout plants of both NQR and AIR12 generated more O 2•- and germinated faster than the single mutant affected in AIR12. To test whether NQR and AIR12 are able to interact functionally, recombinant purified proteins were added to plasma membranes isolated from soybean hypocotyls. In vitro NADH-dependent O 2•- production at the plasma membrane in the presence of NQR was reduced upon addition of AIR12. Electron donation from semi-reduced menadione to AIR12 was shown to take place. Biochemical analysis showed that purified plasma membrane from soybean hypocotyls or roots contained phylloquinone and menaquinone-4 as redox carriers. This is the first report on the occurrence of menaquinone-4 in eukaryotic photosynthetic organisms. We propose that NQR and AIR12 interact via the quinone, allowing an electron transfer from cytosolic NAD(P)H to apoplastic monodehydroascorbate and control thereby the level of reactive oxygen production and the redox state of the apoplast.

Can dicoumarol be used as a gonad-safe anticancer agent: an in vitro and in vivo experimental study.

STUDY HYPOTHESIS: Dicoumarol (DC) has potential for use as a gonad-safe anticancer agent. STUDY FINDING: DC altered cell proliferation, decreased viability and increased apoptosis in Vero and MCF-7 cell lines but did not show any toxic effect on mouse ovarian tissues and developing oocytes in vitro and in vivo. WHAT IS KNOWN ALREADY: DC suppresses cell proliferation and increases apoptosis in various cancer cells such as breast, urogenital and melanoma. DC has also been reported to alter the anticancer effects of several chemotherapeutics, including cisplatin, gemcitabine and doxorubicin in prostate, liver and uroepithelial cancer cells, respectively. STUDY DESIGN, SAMPLES/MATERIALS, METHODS: Vero (African green monkey kidney epithelial cells) and MCF-7 (human cancerous breast epithelial cells) cell lines and mouse granulosa cells isolated from 21-day-old female BALB/c mice (n = 21) were used to assess the effects of DC (10, 50, 100 and 200 µm) for 24 and 48 h on cell proliferation, viability and apoptotic cell death. In vivo experiments were performed with a single i.p. injection of 32 mg/kg DC in 21-day-old female BALB/c mice (n = 12). Following 48 h, animals were sacrificed by cervical dislocation and histological sections of isolated ovaries were evaluated for apoptosis. Viability assays were based on the trypan blue dye exclusion method and an automated cell counter device was used. Terminal deoxynucleotidyltransferase-mediated dUTP nick-end labelling (TUNEL) and Annexin-V immunofluorescence were assessed by 3D confocal microscopy to address apoptotic cell death. We also assessed whether DC inhibits cell proliferation and viability through NQO1 [NAD(P)H Quinone Oxidoreductase 1], an intracellular inhibitor of reactive oxygen species (ROS). The meiotic spindle and chromosomes were studied in mouse oocytes by α-ß-tubulin and 7-aminoactinomycine D (7-AAD) immunostaining in vitro and in vivo. MAIN RESULTS AND THE ROLE OF CHANCE: DC does not block oocyte maturation and no significant alteration was noted in meiotic spindle or chromosome morphology in metaphase-II (M-II) stage oocytes following DC treatment in vitro or in vivo. In contrast, exposure to DC for 24 h suppressed cell proliferation (P = 0.026 at 200 µm), decreased viability (P = 0.002 at 200 µm) and increased apoptosis (P = 0.048 at 100 µm) in Vero and MCF-7 cell lines, compared with controls. These changes were not related to intracellular NQO1 levels. Mouse granulosa cells were unaffected by 50 or 100 µm DC treatment for 24 and 48 h in vitro. DC treatment in vivo did not alter the number of primordial follicles or the ratio of apoptosis in primordial, primary and secondary follicles, as well as in antral follicles, compared with the controls. LIMITATIONS, REASONS FOR CAUTION: DC was tested for ovarian toxicity only in isolated mouse oocytes/ovaries and healthy BALB/c mice. No cancer formation was used as an in vivo test model. The possibility that DC may potentiate ovarian toxicity when combined with traditional chemotherapeutic agents, such as mitomycin-C, cisplatin, gemcitabine and doxorubicin, must be taken into account, as DC is known to alter their effects in some cancer cells. WIDER IMPLICATIONS OF THE FINDINGS: The present study evaluated, for the first time, the effect of DC on ovarian tissue. The results suggested that DC is not toxic to ovarian tissues and developing oocytes; therefore, DC should be assessed further as a potential anticancer agent when female fertility preservation is a concern. LARGE SCALE DATA: N/A. STUDY FUNDING AND COMPETING INTERESTS: This work includes data from dissertation thesis entitled 'Effects of dicoumarol on mitotic and meiotic cells as an anticancer agent' by DA, 2014 and was partly supported by The National Scientific and Technological Research Council of Turkey (SBAG-109S415) to AC, OC and SO. The authors confirm that this article content presents no conflicts of interest.

Characterization of wave phenomena in the relaxation of flash-induced chlorophyll fluorescence yield in cyanobacteria.

Fluorescence yield relaxation following a light pulse was studied in various cyanobacteria under aerobic and microaerobic conditions. In Synechocystis PCC 6803 fluorescence yield decays in a monotonous fashion under aerobic conditions. However, under microaerobic conditions the decay exhibits a wave feature showing a dip at 30-50 ms after the flash followed by a transient rise, reaching maximum at ~1s, before decaying back to the initial level. The wave phenomenon can also be observed under aerobic conditions in cells preilluminated with continuous light. Illumination preconditions cells for the wave phenomenon transiently: for few seconds in Synechocystis PCC 6803, but up to one hour in Thermosynechocystis elongatus BP-1. The wave is eliminated by inhibition of plastoquinone binding either to the QB site of Photosystem-II or the Qo site of cytochrome b6f complex by 3-(3',4'-dichlorophenyl)-1,1-dimethylurea or 2,5-dibromo-3-methyl-6-isopropyl-p-benzoquinone, respectively. The wave is also absent in mutants, which lack either Photosystem-I or the NAD(P)H-quinone oxidoreductase (NDH-1) complex. Monitoring the redox state of the plastoquinone pool revealed that the dip of the fluorescence wave corresponds to transient oxidation, whereas the following rise to re-reduction of the plastoquinone pool. It is concluded that the unusual wave feature of fluorescence yield relaxation reflects transient oxidation of highly reduced plastoquinone pool by Photosystem-I followed by its re-reduction from stromal components via the NDH-1 complex, which is transmitted back to the fluorescence yield modulator primary quinone electron acceptor via charge equilibria. Potential applications of the wave phenomenon in studying photosynthetic and respiratory electron transport are discussed. This article is part of a special issue entitled: photosynthesis research for sustainability: keys to produce clean energy.

NADPH:quinone oxidoreductase 1 regulates host susceptibility to ozone via isoprostane generation.

NADPH:quinone oxidoreductase 1 (NQO1) is recognized as a major susceptibility gene for ozone-induced pulmonary toxicity. In the absence of NQO1 as can occur by genetic mutation, the human airway is protected from harmful effects of ozone. We recently reported that NQO1-null mice are protected from airway hyperresponsiveness and pulmonary inflammation following ozone exposure. However, NQO1 regenerates intracellular antioxidants and therefore should protect the individual from oxidative stress. To explain this paradox, we tested whether in the absence of NQO1 ozone exposure results in increased generation of A(2)-isoprostane, a cyclopentenone isoprostane that blunts inflammation. Using GC-MS, we found that NQO1-null mice had greater lung tissue levels of D(2)- and E(2)-isoprostanes, the precursors of J(2)- and A(2)-isoprostanes, both at base line and following ozone exposure compared with congenic wild-type mice. We confirmed in primary cultures of normal human bronchial epithelial cells that A(2)-isoprostane inhibited ozone-induced NF-κB activation and IL-8 regulation. Furthermore, we determined that A(2)-isoprostane covalently modified the active Cys(179) domain in inhibitory κB kinase in the presence of ozone in vitro, thus establishing the biochemical basis for A(2)-isoprostane inhibition of NF-κB. Our results demonstrate that host factors may regulate pulmonary susceptibility to ozone by regulating the generation of A(2)-isoprostanes in the lung. These observations provide the biochemical basis for the epidemiologic observation that NQO1 regulates pulmonary susceptibility to ozone.

ß-Lapachone ameliorates murine cisplatin nephrotoxicity: NAD⁺, NQO1, and SIRT1 at the crossroads of metabolism, injury, and inflammation.

The clinical utility of cisplatin is limited by nephrotoxicity. Oh et al. report that ß-lapachone prevents this nephrotoxicity but not cisplatin's cytotoxicity for cancers. In addition to its potential clinical importance, the beneficial effect of ß-lapachone on cisplatin acute kidney injury may illustrate fundamental processes that ordinarily link alterations in nutrient availability and intracellular reactive oxygen species on the one hand, with inflammation and cell death on the other hand.

Pharmacological activation of NQO1 increases NAD⁺ levels and attenuates cisplatin-mediated acute kidney injury in mice.

Cisplatin is a widely used chemotherapeutic agent for the treatment of various tumors. In addition to its antitumor activity, cisplatin affects normal cells and may induce adverse effects, such as ototoxicity, nephrotoxicity, and neuropathy. Various mechanisms, such as DNA adduct formation, mitochondrial dysfunction, oxidative stress, and inflammatory responses, are critically involved in cisplatin-induced adverse effects. As NAD(+) is a cofactor for various enzymes associated with cellular homeostasis, we studied the effects of increased NAD(+) levels by means of NAD(P)H: quinone oxidoreductase 1 (NQO1) activation using a known pharmacological activator (ß-lapachone) in wild-type and NQO1(-/-) mice on cisplatin-induced renal dysfunction in vivo. The intracellular NAD(+)/NADH ratio in renal tissues was significantly increased in wild-type mice co-treated with cisplatin and ß-lapachone compared with the ratio in mice treated with cisplatin alone. Inflammatory cytokines and biochemical markers for renal damage were significantly attenuated by ß-lapachone co-treatment compared with those in the cisplatin alone group. Notably, the protective effects of ß-lapachone in wild-type mice were completely abrogated in NQO1(-/-) mice. Moreover, ß-lapachone enhanced the tumoricidal action of cisplatin in a xenograft tumor model. Thus, intracellular regulation of NAD(+) levels through NQO1 activation might be a promising therapeutic target for the protection of cisplatin-induced acute kidney injury.

Quinone-induced protein handling changes: implications for major protein handling systems in quinone-mediated toxicity.

Para-quinones such as 1,4-Benzoquinone (BQ) and menadione (MD) and ortho-quinones including the oxidation products of catecholamines, are derived from xenobiotics as well as endogenous molecules. The effects of quinones on major protein handling systems in cells; the 20/26S proteasome, the ER stress response, autophagy, chaperone proteins and aggresome formation, have not been investigated in a systematic manner. Both BQ and aminochrome (AC) inhibited proteasomal activity and activated the ER stress response and autophagy in rat dopaminergic N27 cells. AC also induced aggresome formation while MD had little effect on any protein handling systems in N27 cells. The effect of NQO1 on quinone induced protein handling changes and toxicity was examined using N27 cells stably transfected with NQO1 to generate an isogenic NQO1-overexpressing line. NQO1 protected against BQ-induced apoptosis but led to a potentiation of AC- and MD-induced apoptosis. Modulation of quinone-induced apoptosis in N27 and NQO1-overexpressing cells correlated only with changes in the ER stress response and not with changes in other protein handling systems. These data suggested that NQO1 modulated the ER stress response to potentiate toxicity of AC and MD, but protected against BQ toxicity. We further demonstrated that NQO1 mediated reduction to unstable hydroquinones and subsequent redox cycling was important for the activation of the ER stress response and toxicity for both AC and MD. In summary, our data demonstrate that quinone-specific changes in protein handling are evident in N27 cells and the induction of the ER stress response is associated with quinone-mediated toxicity.

NAD(P)H quinone oxidoreductase 1 (NQO1)-bioactivated pronqodine A regulates prostaglandin release from human synovial sarcoma cells.

Natural products have contributed to the elucidation of biological mechanisms as well as drug discovery research. Even now, the expectation for natural products is undiminished. We screened prostaglandin release inhibitors that had no effect on in vitro cyclooxygenase activity derived from natural product sources and discovered pronqodine A. Using spectral analysis and total synthesis, the structure of pronqodine A was shown to be a benzo[d]isothiazole-4,7-dione analogue. Evaluation of the biological activity of pronqodine A revealed that the NAD(P)H dehydrogenase quinone 1 (NQO1) converted pronqodine A into a two-electron reductive form. The reductive form underwent autoxidation and reversed to its native form immediately with the generation of reactive oxygen species. Further investigations proved that pronqodine A inhibited cyclooxygenase enzyme activity only in the presence of NQO1. Pronqodine A acts as a potential bioreductive compound, inhibiting prostaglandin release in selectively activated NQO1-expressing cells.

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.

Plasma membrane electron transport in pancreatic ß-cells is mediated in part by NQO1.

Plasma membrane electron transport (PMET), a cytosolic/plasma membrane analog of mitochondrial electron transport, is a ubiquitous system of cytosolic and plasma membrane oxidoreductases that oxidizes cytosolic NADH and NADPH and passes electrons to extracellular targets. While PMET has been shown to play an important role in a variety of cell types, no studies exist to evaluate its function in insulin-secreting cells. Here we demonstrate the presence of robust PMET activity in primary islets and clonal ß-cells, as assessed by the reduction of the plasma membrane-impermeable dyes WST-1 and ferricyanide. Because the degree of metabolic function of ß-cells (reflected by the level of insulin output) increases in a glucose-dependent manner between 4 and 10 mM glucose, PMET was evaluated under these conditions. PMET activity was present at 4 mM glucose and was further stimulated at 10 mM glucose. PMET activity at 10 mM glucose was inhibited by the application of the flavoprotein inhibitor diphenylene iodonium and various antioxidants. Overexpression of cytosolic NAD(P)H-quinone oxidoreductase (NQO1) increased PMET activity in the presence of 10 mM glucose while inhibition of NQO1 by its inhibitor dicoumarol abolished this activity. Mitochondrial inhibitors rotenone, antimycin A, and potassium cyanide elevated PMET activity. Regardless of glucose levels, PMET activity was greatly enhanced by the application of aminooxyacetate, an inhibitor of the malate-aspartate shuttle. We propose a model for the role of PMET as a regulator of glycolytic flux and an important component of the metabolic machinery in ß-cells.

Role for NAD(P)H:quinone oxidoreductase 1 and manganese-dependent superoxide dismutase in 17-(allylamino)-17-demethoxygeldanamycin-induced heat shock protein 90 inhibition in pancreatic cancer cells.

Previous work demonstrated that NAD(P)H:quinone oxidoreductase 1 (NQO1) metabolized the heat shock protein 90 (Hsp90) inhibitor 17-(allylamino)-17-demethoxygeldanamycin (17AAG) to the corresponding hydroquinone (17AAGH2). The formation of 17AAGH2 by NQO1 results in a molecule that binds with greater affinity to Hsp90 compared with the parent quinone. 17AAG induced substantial growth inhibition in human pancreatic cancer cell lines expressing NQO1. Growth inhibition induced by 17AAG could be reduced by pretreatment with 5-methoxy-1,2-dimethyl-3-[(4-nitrophenoxy)methyl]-indole-4,7-dione (ES936), a mechanism-based inhibitor of NQO1. After treatment with 17AAG, biomarkers of Hsp90 inhibition, including markers of cell-cycle arrest, were more pronounced in NQO1-expressing cells compared with NQO1-null cells. The intracellular concentrations of 17AAG and 17AAGH2 were measured in human pancreatic cancer cells, and it was observed that larger amounts of 17AAG and 17AAGH2 could be detected in cells with catalytically active NQO1 compared with cells lacking NQO1 activity or cells pretreated with ES936. These data demonstrate that, in addition to generating an inhibitor with greater affinity for Hsp90 (17AAGH2), reduction of 17AAG to 17AAGH2 by NQO1 leads to substantially greater intracellular concentrations of 17AAG and 17AAGH2. In addition, oxidation of 17AAGH2 could be prevented by superoxide dismutase (SOD), demonstrating that 17AAGH2 was sensitive to oxidation by superoxide. Stable transfection of manganese-dependent SOD into MiaPaCa-2 cells resulted in a significantly greater intracellular concentration of 17AAGH2 with a corresponding increase in growth inhibitory activity. These data confirm the role of NQO1 in sensitivity to 17AAG and demonstrate that SOD functions in conjunction with NQO1 to maintain intracellular levels of 17AAGH2, the active Hsp90 inhibitor derived from 17AAG.

Comparison of activation of aristolochic acid I and II with NADPH:quinone oxidoreductase, sulphotransferases and N-acetyltranferases.

OBJECTIVES: Ingestion of aristolochic acid (AA) is associated with development of urothelial tumors linked with aristolochic acid nephropathy, and is implicated in the development of Balkan endemic nephropathy-associated urothelial tumors. Aristolochic acid I (AAI), the major toxic component of AA, is more toxic than its demethoxylated derivate AAII. A different enzymatic conversion of both carcinogens might be one of the reasons explaining this feature. Therefore, the present study has been designed to compare efficiency of human NAD(P)H:quinone oxidoreductase (NQO1) and phase II enzymes such as sulfotransferases (SULTs) and N,O-acetyltransferases (NATs) to activate AAI and AAII in vitro. In addition, to investigate the molecular mechanisms of AAI and AAII reduction by human NQO1, molecular modeling was used to compare interactions of AAI and AAII with the active site of this enzyme. METHODS: DNA adduct formation by AAI and AAII was investigated by the nuclease P1 version of the 32P-postlabeling method. In silico docking, employing soft-soft (flexible) docking procedure, was used to study the interactions of AAI and AAII with the active site of human NQO1. RESULTS: Human NQO1 activated AAI and AAII, generating DNA adduct patterns reproducing those found in several species including human exposed to these compounds. These results demonstrate that NQO1 is capable of reducing both AAs to reactive species binding to DNA. However, concentrations required for half-maximum DNA binding mediated by NQO1 were higher for AAII (158 µM) than for AAI (17 µM). One of the reasons causing this phenomenon is a lower efficiency of NQO1 to reduce AAII than AAI we found in this work; although both AAI and AAII are bound with similar binding affinities to the NQO1 active site, the binding orientation of AAII in the active site of NQO1 does not favor the effective reduction of its nitro group. Because reduced nitro-aromatics are often further activated by SULTs or NATs, their roles in AAI and AAII activation were investigated. Our results indicate that phase II reactions do not stimulate the bioactivation of AAs; neither enzymes present in human hepatic cytosols nor human SULT1A1, 1A2, 1A3, 1E, or 2A nor NAT1 or NAT2 further enhanced DNA adduct formation by AAs. In contrast, human SULT1A1, 1A2 and 1A3 as well as NAT1 and NAT2 enzymes even inhibited NQO1-mediated bioactivation of AAII. Therefore, under the in vitro conditions used, DNA adducts arise by enzymatic reduction of AAs through the formation of N-hydroxyaristolactams that are spontaneously decomposed to the reactive species forming DNA adducts. CONCLUSION: The results found in this study emphasize the importance of NQO1 in the metabolic activation of AAI and AAII and provide the evidence that initial nitroreduction is the rate limiting step in their activation. This enzyme is more effective in activation of AAI relative to AAII, which might contribute to its lower binding to DNA found both in vitro and in vivo, Moreover, inhibition effects of conjugation reactions on AAII activation might further contribute to its decreased capability of forming DNA adducts and its lower toxicity comparing with AAI.

The redox-sensing quinone reductase Lot6p acts as an inducer of yeast apoptosis.

Mammalian NAD(P)H:quinone oxidoreductases such as human NQO1 act as inducers of apoptosis. Quinone reductases generated interest over the last decade due to their proposed function in the oxidative stress response. Furthermore, human NQO1 was reported to regulate p53 stability and p53-dependent apoptosis through regulation of cellular oxidation-reduction events. In this study, we have used low concentrations of hydrogen peroxide (0.4 and 0.6 mM) to induce apoptosis-like cell death in wild type, an LOT6 overexpressing and a Deltalot6 yeast strain to monitor cell survival. Using this approach, we demonstrate that yeast quinone reductase Lot6p, an orthologue of mammalian quinone reductases, plays a pivotal role in apoptosis-like cell death in Saccharomyces cerevisiae. Overexpression of LOT6 results in enhanced cell death, as shown by an investigation of the morphological hallmarks of apoptosis-like fragmentation of DNA and externalization of phosphatidylserine, whereas the deletion strain displays a deficiency in apoptosis-like cell death as compared with the wild type. Thus, we propose that Lot6p is directly involved in the control of the apoptosis-like cell death in yeast.

Role of Nrf2-antioxidant in radioprotection by root extract of Inula racemosa.

Purpose:Inula racemosa, a Trans-Himalayan plant is an important medicinal herb. In this study, the radio-modulatory efficacy of aqueous root extract of I. racemosa was investigated. Materials and methods: Normal Kidney Epithelium cells were treated with extract (50-200 µg/ml) and exposed to 3 Gy of γ-radiation, while C57BL/6 mice were administered with extract (300-600 mg/kg BW) intraperitoneally prior to exposure to 7.5 Gy of γ-radiation to assess radiation modulatory efficacy. Results: The administration of extract (30 min and 1 h) prior to radiation exposure improved the survival of NKE cells (as measured by proliferation), restored MMP and ROS levels as compared to radiation-exposed alone cells. These cells showed up-regulated Nrf2 protein levels at 7 h and increased expression of HO-1 and NOQ1 protein at 24 h In mice, the 30 days whole body survival study demonstrated that extract pre-treatment increases survival or delays the onset of radiation-induced mortality as against 70% mortality of 7.5 Gy of γ-radiation. Conclusions: The aqueous extract of roots of I. racemosa enhanced the survival of irradiated NKE cells and rescued C57BL/6 mice against WBI-induced mortality. The radiation modulation efficacy was mediated through cumulative activation of HO-1 and NQO1 downstream of Nrf2 translocation in NKE cells. Abbreviations: ARE: Antioxidant Response Element; FITC: Fluorescein isothiocyanate; GCS: Glutamylcysteine synthase; HO-1: Heme oxygenase-1; LPS: Lipopolysacharide; MRP: Multidrug Resistance-Associated Proteins; NQO1: NAD(P)H Quinone Dehydrogenase 1; NRH: Quinone Oxidoreductase 2 (NQO2); PBS: Phosphate Buffer Saline; PKA: Protein Kinase A; PKC: Protein Kinase C; PI3-kinase: Phosphatidylinositol 3-Kinase; SRB: Sulforhodamine B; UV: Ultra-Violet radiation.

Amygdalin attenuates acute liver injury induced by D-galactosamine and lipopolysaccharide by regulating the NLRP3, NF-κB and Nrf2/NQO1 signalling pathways.

Acute liver injury (ALI) is a life-threatening syndrome accompanied by overwhelming inflammation. Amygdalin (AGD) has been reported to possess various biological activities, particularly anti-inflammatory activity. The current study was designed to assess the protective effects and underlying mechanisms of AGD against ALI induced by d-galactosamine (GalN) and lipopolysaccharide (LPS) in mice. The results indicated that AGD treatment effectively reduced the lethality, ameliorated the histopathological liver changes, reduced the malondialdehyde (MDA) and myeloperoxidase (MPO) levels, and decreased the alanine transaminase (ALT) and aspartate aminotransferase (AST) levels resulting from LPS/GalN challenge. Moreover, AGD significantly inhibited LPS/GalN-induced inflammatory responses in mice with ALI by reducing not only the secretion of tumour necrosis factor (TNF)-α, interleukin (IL)-1ß, and IL-6 but also the protein expression of inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2). Additionally, our results demonstrated that the inhibitory effect of AGD was due to the suppressed activation of nuclear factor-kappa B (NF-κB) and nucleotide-binding domain (NOD-)like receptor protein 3 (NLRP3) inflammasome activity. Furthermore, AGD treatment substantially increased nuclear factor erythroid 2-related factor 2 (Nrf2) nuclear translocation and enhanced NAD (P) H: quinoneoxidoreductase 1 protein expression, which was reversed by a Nrf2 inhibitor, in HepG2 cells. In summary, our investigations suggested that the ability of AGD to ameliorate LPS/GalN-induced ALI may involve the inhibition of the NLRP3 inflammasome and NF-κB signalling pathways and the upregulation of the Nrf2/NQO1 signalling pathway.

Protective roles of quinone reductase and tamoxifen against estrogen-induced mammary tumorigenesis.

We previously reported that antiestrogen-liganded estrogen receptor beta (ERbeta) transcriptionally activates the major detoxifying enzyme quinone reductase (QR) (NAD(P)H:quinone oxidoreductase). Further studies on the functional role of ERbeta-mediated upregulation of antioxidative enzymes indicated protective effects against estrogen-induced oxidative DNA damage (ODD). We now report on in vivo and in vitro studies that show that ERbeta-mediated upregulation of QR are involved in the protection against estrogen-induced mammary tumorigenesis. Using the August Copenhagen Irish (ACI) model of estrogen-induced carcinogenesis, we observed that increased ODD and decreased QR expression occur early in the process of estrogen-induced mammary tumorigenesis. Prevention of ACI mammary gland tumorigenesis by tamoxifen was accompanied by decreased ODD and increased QR levels. These correlative findings were supported by our findings that downregulation of QR levels led to increased levels of estrogen quinone metabolites and enhanced transformation potential of 17beta-estradiol treated MCF10A non-tumorigenic breast epithelial cells. Concurrent expression of ERbeta and treatment with 4-hydroxytamoxifen decreased tumorigenic potential of these MCF10A cells. We conclude that upregulation of QR, through induction by tamoxifen, can inhibit estrogen-induced ODD and mammary cell tumorigenesis, representing a possible novel mechanism of tamoxifen prevention against breast cancer.

Control of cellular redox status and upregulation of quinone reductase NQO1 via Nrf2 activation by alpha-lipoic acid in human leukemia HL-60 cells.

alpha-Lipoic acid (LA) is a naturally-occurring micronutrient that has been actively investigated for the treatment and management of various chronic medical conditions including neurodegenerative diseases, diabetes and hepatic disorders. However, relatively few studies have examined the effects of LA as a chemopreventive agent, particularly in regard to its ability to modulate homeostasis of oxidoreductive state and to regulate detoxification enzymes such as quinone reductase NQO1 in LA-responsive cells. We tested the hypothesis that LA affects the intracellular redox status and induces NQO1 expression using the human promyelocytic HL-60 leukemia cells. We showed that treatment by LA maintains HL-60 cells in a relatively reduced state, supported by the dose/time-dependent increase in the activities of glutathione peroxidase and glutathione reductase and decrease in the activity of catalase. Moreover, LA significantly increased the activity and protein expression of NQO1. The induction of NQO1 was accompanied by the nuclear accumulation of transcription factor Nrf2, which was correlated with a decreased level of Nrf2 in the cytosol as well as the concomitant reduction in the expression of cytoplasmic repressor of Nrf2, Keap1.

The multiplicity of dehydrogenases in the electron transport chain of plant mitochondria.

The electron transport chain in mitochondria of different organisms contains a mixture of common and specialised components. The specialised enzymes form branches to the universal electron path, especially at the level of ubiquinone, and allow the chain to adjust to different cellular and metabolic requirements. In plants, specialised components have been known for a long time. However, recently, the known number of plant respiratory chain dehydrogenases has increased, including both components specific to plants and those with mammalian counterparts. This review will highlight the novel branches and their consequences for the understanding of electron transport and redundancy of electron paths.