H-quinone Oxidoreductase Research Articles

Naringenin Ameliorates Behavioral Dysfunction and Neurological Deficits in a d-Galactose-Induced Aging Mouse Model Through Activation of PI3K/Akt/Nrf2 Pathway.

In this study, we evaluated the protective effects of naringenin on aging mice induced by d-galactose (d-gal). Open field test and Morris water maze test were performed to evaluate the effect of naringenin on behavioral dysfunction. Hematoxylin-eosin staining, TUNEL staining, and Nissl staining were used to estimate the effect of naringenin on neurological deficits. Furthermore, naringenin markedly activated PI3K/Akt signaling, eventually promoted the nuclear translocation of nuclear factor-erythroid 2-related factor 2, and induced the expression of heme oxygenase 1 and NAD(P)H-quinone oxidoreductase 1. Superoxide dismutase (SOD), catalase (CAT), and thiobarbituric acid reactive substances (TBARS) assays were conducted to evaluate oxidative stress in d-gal-induced aging mice. Our finding demonstrated that naringenin was a promising agent for attenuating the aging process, and enhancing endogenous antioxidant defense capacity was a reliable strategy to delay the senescence process.

The time-dependent effects of St John’s wort on cytochrome P450, uridine diphosphate-glucuronosyltransferase, glutathione S-transferase, and NAD(P)H-quinone oxidoreductase in mice

Hypericum perforatum [St. John’s wort (SJW)] is known to cause a drug interaction with the substrates of cytochrome P450 (P450, CYP) isoforms, mainly CYP3A. This study aims to determine the dose response and time course of the effects of SJW extract on P450s, UDP-glucuronosyltransferase (UGT), glutathione S-transferase (GST), and NAD(P)H-quinone oxidoreductase (NQO) in mice. The oral administration of SJW extract to male mice at 0.6 g/kg/d for 21 days increased hepatic oxidation activity toward a Cyp3a substrate nifedipine. By extending the SJW treatment to 28 days, hepatic nifedipine oxidation (NFO) and warfarin 7-hydroxylation (WOH) (Cyp2c) activities were increased by 95% and 34%, respectively. Immunoblot analysis of liver microsomal proteins revealed that the Cyp2c protein level was elevated by the 28-day treatment. However, the liver microsomal activities of the oxidation of the respective substrates of Cyp1a, Cyp2a, Cyp2b, Cyp2d, and Cyp2e1 remained unchanged. In the kidney, SJW increased the NFO, but not the WOH activity. The extended 28-day treatment did not alter mouse hepatic and renal UGT, GST, and NQO activities. These findings demonstrate that SJW stimulates hepatic and renal Cyp3a activity and hepatic Cyp2c activity and expression. The induction of hepatic Cyp2c requires repeated treatment for a period longer than the initial induction of Cyp3a.

Roles of NAD (P) H-Quinone Oxidoreductase 1 (NQO1) On Cancer Progression and Chemoresistance

NAD(P)H-Quinone Oxidoreductase 1 (NQO1), originally referred to as DT-diaphorase, is a xenobiotic metabolizing/antioxidant enzyme that detoxifies chemical stressors, providing cytoprotection in normal tissues. NQO1 catalyzes obligatory two-electron reduction of several endogenous and environmental quinones to hydroquinone that are ready for further conjugation and excretion. The enzyme requires NADH or NADPH as an electron donor for enzymatic activity. High-level of NQO1 expression has however, been correlated with numerous human malignancies, suggesting its role in cancer progression and chemoresistance. This adaptation renders the cancer cells to survive in relatively high oxidative stress condition compared to normal cells, as well as protects cancer cells from toxic action of chemotherapeutic agents. Inhibitors of NQO1 enzyme have been found to improve anticancer activities of conventional chemotherapeutic agents. The review provides a perspective on a molecular basis of NQO1-mediated cancer cells progression and the suppression and possible strategy to improve chemosensitivity and to overcome chemoresistance of NQO1 inhibitors when used in combination with chemotherapeutic drugs.

Alkynyloxy derivatives of 5,8-quinolinedione: Synthesis, in vitro cytotoxicity studies and computational molecular modeling with NAD(P)H:Quinone oxidoreductase 1

The natural 7-amino-5,8-quinolinodione antibiotics were the substrate for the NQO1 protein. The structure-activity relationship showed that the 5,8-quinolinedione moiety was responsible for the interaction with the enzyme. In our research, we presented the synthesis, cytotoxic activity and theoretical study of a 5,8-quinolinedione compound as a potential inhibitor of the NQO1 enzyme. Mono and disubstituted alkynyloxy derivatives of the 5,8-quinolinedione were synthesized and characterized by 1H, 13C NMR, IR and HR-MS spectra. Newly synthesized derivatives were also tested for their biological activity in vitro against the human cancer cell lines. They showed high cytotoxic activity depending on the type of substituent and the employed tumor cell lines. Moreover, two breast cancer cell lines, MDA-MB-231 and MCF-7, were chosen for analysis as they are characterized by different NQO1 protein levels. It was found that cytotoxic activities of all studied compounds increased against the cell line with a higher NQO1 protein level. The molecular docking was used to examine the probable interaction between the molecules of alkynyloxy derivatives and human NAD[P]H-quinone oxidoreductase (NQO1) protein. The computational studies showed that the 5,8-quinolinedione moiety of all docked compounds was located deeply in the hydrophobic matrix of the active site near the side chains of aromatic residues in positions Trp 105, Phe 178, Tyr 126 and Tyr 128. In every case, introduction of aromatic moieties into the 5,8-quinolinedione molecule led to an increase in the binding affinity in relation to the 6,7-dichloro-5,8-quinolinedione.

Miltirone protects human EA.hy926 endothelial cells from oxidized low-density lipoprotein-derived oxidative stress via a heme oxygenase-1 and MAPK/Nrf2 dependent pathway

BackgroundOxidized low-density lipoprotein (ox-LDL) is an underlying cause of endothelial dysfunction, which is an early event in the pathogenesis of atherosclerosis. In our previous study, we established an ARE-driven luciferase reporter system and screened out several potential Nrf2 activators from Salvia miltiorrhiza Bunge. PurposeSince miltirone showed the most potent ARE-driven luciferase activity, the aim of this study was to test the protective role of miltirone against oxidative stress in endothelial cell and to investigate the underlying mechanistic signaling pathways. Study Design/MethodIn the present study, miltirone increased the expression of nuclear translocation and transcriptional activities of NF-E2-related factor 2 (Nrf2), which led to augmented expression of antioxidant-response element (ARE)-dependent heme oxygenase-1 (HO-1) and NAD(P)H-quinone oxidoreductase 1 (NQO1). Inhibition of Nrf2/HO-1 by RNA interference abolished miltirone-induced cytoprotective effects against ox-LDL, which suggested that Nrf2 and the downstream expression of HO-1 are required for the functional effects of miltirone. Ox-LDL-stimulated mitogen-activated protein kinase activation, ROS production, and miltirone dramatically inhibited synthesis of ROS, as well as decreased SOD and glutathione S-transferase (GST) in human EA.hy926 endothelial cells. ResultsMiltirone-induced Nrf2 and HO-1 expression was related to mitogen-activated protein kinase (MAPK) pathways. The activation of MAPK was partially dependent on the phosphorylation of the c-Jun NH2-terminal kinase (JNK) and extracellular signal-regulated kinase (ERK) pathways, but not P38 MAPK signaling. However, miltirone-induced Nrf2/HO-1 expression can only be effectively blocked by JNK inhibitor SP600125. ConclusionOur findings reveal that miltirone exerts protective functions on endothelial cells in response to ox-LDL-induced oxidative stress, and does so via Nrf2/HO-1, which provides novel insights into the antioxidant capacity of miltirone.

Salidroside's Protection Against UVB-Mediated Oxidative Damage and Apoptosis Is Associated with the Upregulation of Nrf2 Expression.

Salidroside is the major active component of Rhodiola rosea, a traditional Chinese herbal medicine used for protection against ultraviolet (UV) radiation. This study investigated whether salidroside can protect skin from ultraviolet B (UVB)-induced oxidative damage in human immortalized HaCaT keratinocytes and the skin of guinea pigs. Using HaCaT cell models, the effects of salidroside on oxidative damage and possible regulatory factors [including NF-E2-related factor 2 (Nrf2), NAD(P)H-quinone oxidoreductase (NQO1), and heme oxygenase 1 (HO-1)] were examined. In addition, the regulatory effects of salidroside on apoptotic sunburn cells (SBCs) and 8-hydroxy-2'-deoxyguanosine (8-OHdG)-positive epidermal cells on UVB-exposed guinea pig skin were also investigated. We found that salidroside pretreatment upregulated Nrf2 translocation to the nucleus and transcription activity in HaCaT cells, as reflected by the increased nuclear accumulation of Nrf2 as well as the gene and protein expression of downstream Nrf2 antioxidants, including NQO1 and HO-1. In addition, we also found that pretreatment with salidroside reactive oxygen species (ROS) in irradiated HaCaT cells. The oral administration of salidroside (0.1% w/w) to guinea pigs inhibited the UVB-mediated formation of apoptotic SBCs and 8-OHdG-positive epidermal cells in the skin of guinea pigs. Our results show that UVB-induced oxidative damage can be prevented by salidroside with upregulation of nuclear Nrf2 expression.

NAD(P)H-dependent quinone oxidoreductase 1 (NQO1) and cytochrome P450 oxidoreductase (CYP450OR) differentially regulate menadione-mediated alterations in redox status, survival and metabolism in pancreatic β-cells

NQO1 (NAD(P)H-quinone oxidoreductase 1) reduces quinones and xenobiotics to less-reactive compounds via 2-electron reduction, one feature responsible for the role of NQO1 in antioxidant defense in several tissues. In contrast, NADPH cytochrome P450 oxidoreductase (CYP450OR), catalyzes the 1-electron reduction of quinones and xenobiotics, resulting in enhanced superoxide formation. However, to date, the roles of NQO1 and CYP450OR in pancreatic β-cell metabolism under basal conditions and oxidant challenge have not been characterized. Using NQO1 inhibition, over-expression and knock out, we have demonstrated that, in addition to protection of β-cells from toxic concentrations of the redox cycling quinone menadione, NQO1 also regulates the basal level of reduced-to-oxidized nucleotides, suggesting other role(s) beside that of an antioxidant enzyme. In contrast, over-expression of NADPH cytochrome P450 oxidoreductase (CYP450OR) resulted in enhanced redox cycling activity and decreased cellular viability, consistent with the enhanced generation of superoxide and H2O2. Basal expression of NQO1 and CYP450OR was comparable in isolated islets and liver. However, NQO1, but not CYP450OR, was strongly induced in β-cells exposed to menadione. NQO1 and CYP450OR exhibited a reciprocal preference for reducing equivalents in β-cells: while CYP450OR preferentially utilized NADPH, NQO1 primarily utilized NADH. Together, these results demonstrate that NQO1 and CYP450OR reciprocally regulate oxidant metabolism in pancreatic β-cells.

Effect of rutin and hesperidin on the expression of Nrf2 gene and the activity of hemoxygenase-1 and NAD(P)H-quinone oxidoreductase at their separate and combined action

Flavonoids rutin (R) and hesperidin (Hes) have a broad spectrum of the biological activity based on their antiradical properties and ability to increase the activity of antioxidant enzymes, including the activity of heme oxygenase-1 (HO-1) and NAD(P)H-quinone oxidoreductase (QR). It is supposed that the main regulator of the activity of HO-1 and QR is the transcription factor Nrf2. The purpose of the study was to determine the effects of R and Hes on the expression of Nrf2 gene and protein, on the activity and mRNA and protein expression of HO-1 and QR at their separate and combined action. Administration in diet of male Wistar rats (with initial body weight 180-200 g) R (400 mg/kg b.w.) and Hes (400 mg/ kg b.w.) during 14 days separately or in combination had no toxic or pro-oxidant effect, which were assessed by the level of liver MDA, hydroperoxides of lipids, reduced and oxidized glutathione. R and, to a lesser extent, Hes caused increased activity of HO-1 and QR. Their combined effect on the activity of HO-1 did not differ from the separate effect of each flavonoid. The combined action of R and Hes on the activity of QR was additive. According to Western blotting, changes in HO-1, Nrf2 and QR protein levels under the action of R and Hes separately or in combination were not statistically significant. The results of real time PCR demonstrated the presence of small, but statistically significant, changes in the level of expression of the genes Nrf2 (Nrf2), HO-1 (Hmox1) and QR (NQO1) both for separate and combined action of R and Hes. Thus, the obtained results showed that high-dose of R and Hes separately and in combination didn't significantly affect the gene and protein expression of transcription factor Nrf2 and that the increased activity of HO-1 and QR was not associated with the increased expression of Hmox1 and NQO1 genes.

Liver expression of Nrf2-related genes in different liver diseases

The KEAP1-Nrf2 antioxidant signaling pathway is important in protecting liver from various insults. However, little is known about the expression of Nrf2-related genes in human liver in different diseases. This study utilized normal donor liver tissues (n=35), samples from patients with hepatocellular carcinoma (HCC, n=24), HBV-related cirrhosis (n=27), alcoholic cirrhosis (n=5) and end-stage liver disease (n=13). All of the liver tissues were from the Oriental Liver Transplant Center, Beijing, China. The expressions of Nrf2 and Nrf2-related genes, including its negative regulator Kelch-like ECH-associated protein 1 (KEAP1), its targeted gene NAD(P)H-quinone oxidoreductase 1 (NQO1), glutamate-cysteine ligase catalytic subunit (GCLC) and modified subunit (GCLM), heme oxygenase 1 (HO-1) and peroxiredoxin-1 (PRDX1) were evaluated. The expression of Nrf2 was decreased in HCC, increased in alcoholic cirrhosis and end-stage liver disease. The expression of KEAP1 was increased in all of the liver samples. The most notable finding was the increased expression of NQO1 in HCC (18-fold), alcoholic cirrhosis (6-fold), end-stage liver disease (5-fold) and HBV-related cirrhosis (3-fold). Peri-HCC also had 4-fold higher NQO1 mRNA as compared to the normal livers. GCLC mRNA levels were lower only in HCC, as compared to the normal livers and peri-HCC tissues. GCLM mRNA levels were higher in HBV-related cirrhosis and end-stage liver disease. HO-1 mRNA levels were increased in all liver tissues except for HCC. Peri-HCC had higher PRDX1 mRNA levels compared with HCC and normal livers. Nrf2 and Nrf2-related genes are aberrantly expressed in the liver in different diseases and the increase of NQO1 was the most notable finding, especially in HCC.

Melatonin induces the expression of Nrf2-regulated antioxidant enzymes via PKC and Ca2+ influx activation in mouse pancreatic acinar cells

The goal of this study was to evaluate the potential activation of the nuclear factor erythroid 2-related factor and the antioxidant-responsive element (Nrf2–ARE) signaling pathway in response to melatonin in isolated mouse pancreatic acinar cells. Changes in intracellular free Ca2+ concentration were followed by fluorimetric analysis of fura-2-loaded cells. The activations of PKC and JNK were measured by Western blot analysis. Quantitative reverse transcription–polymerase chain reaction was employed to detect the expression of Nrf2-regulated antioxidant enzymes. Immunocytochemistry was employed to determine nuclear location of phosphorylated Nrf2, and the cellular redox state was monitored following MitoSOX Red-derived fluorescence. Our results show that stimulation of fura-2-loaded cells with melatonin (1µM to 1mM), in the presence of Ca2+ in the extracellular medium, induced a slow and progressive increase of [Ca2+]c toward a stable level. Melatonin did not inhibit the typical Ca2+ response induced by CCK-8 (1nM). When the cells were challenged with indoleamine in the absence of Ca2+ in the extracellular solution (medium containing 0.5mM EGTA) or in the presence of 1mM LaCl3, to inhibit Ca2+ entry, we could not detect any change in [Ca2+]c. Nevertheless, CCK-8 (1nM) was able to induce the typical mobilization of Ca2+. When the cells were incubated with the PKC activator PMA (1µM) in the presence of Ca2+ in the extracellular medium, we observed a response similar to that noted when the cells were challenged with melatonin 100µM. However, in the presence of Ro31-8220 (3µM), a PKC inhibitor, stimulation of cells with melatonin failed to evoke changes in [Ca2+]c. Immunoblots, using an antibody specific for phospho-PKC, revealed that melatonin induces PKCα activation, either in the presence or in the absence of external Ca2+. Melatonin induced the phosphorylation and nuclear translocation of the transcription factor Nrf2, and evoked a concentration-dependent increase in the expression of the antioxidant enzymes NAD(P)H-quinone oxidoreductase 1, catalytic subunit of glutamate-cysteine ligase, and heme oxygenase-1. Incubation of MitoSOX Red-loaded pancreatic acinar cells in the presence of 1nM CCK-8 induced a statistically significant increase in dye-derived fluorescence, reflecting an increase in oxidation, that was abolished by pretreatment of cells with melatonin (100µM) or PMA (1µM). On the contrary, pretreatment with Ro31-8220 (3µM) blocked the effect of melatonin on CCK-8-induced increase in oxidation. Finally, phosphorylation of JNK in the presence of CCK-8 or melatonin was also observed. We conclude that melatonin, via modulation of PKC and Ca2+ signaling, could potentially stimulate the Nrf2-mediated antioxidant response in mouse pancreatic acinar cells.

АФК-генеруюча та антиоксидантна системи печінки щурів за дії преднізолону і вітаміну D3

The mechanisms of glucocorticoid-induced disturbances of liver function is currently not fully clarified. Vitamin D3 was previously shown to play an important role in the regulation of impaired oxidative metabolism and detoxification function of the liver associated with the effects of hepatotoxic compounds. The study was undertaken to define the intensity of oxidative metabolism in the rat liver and survival of hepatocytes after prolonged prednisolone administration and to assess whether vitamin D3 is capable to counter glucocorticoid-induced changes. It has been shown that prednisolone (0.5 mg per animal for 30 days) leads to 1.6-fold increase in the percentage of necrotic cells among isolated hepatocytes as compared with the control. The glucocorticoid-induced impairment of hepatocellular function was accompanied by enhanced generation of reactive oxygen species (ROS), accumulation of TBA-active products and carbonylated proteins but reduced levels of free SH-groups of low molecular weight compounds. It was demonstrated a decrease in the activities of key enzymes of antioxidant system (SOD, catalase, glutathione peroxidase), whereas the activities of pro-oxidant enzymes NAD(P)H-quinone oxidoreductase and semicarbazide-sensitive amine oxidase were shown to be increased. Vitamin D3 (and to greater extent in combination with α-tocopherol) administration (100 IU) on the background of glucocorticoid therapy caused normalizing effects on the level of ROS formation, oxidative modification of biomolecules and activity of antioxidant enzymes resulting in better survival of hepatocytes. These data suggest a potential role of vitamin D3 in the regulation of oxidative metabolism alterations related to hepatotoxic action of glucocorticoids.

Association of NQO1 and TNF polymorphisms with Parkinson's disease: A meta-analysis of 15 genetic association studies.

Parkinson's disease (PD) is a neurodegenerative movement d'isorder that affects ~2% of the population aged ≥65 years. NAD(P)H-quinone oxidoreductase 1 (NQO1) and tumor necrosis factor-α (TNF-α) are two important factors in the generation of oxidative stress in PD. The aim of the present study was to assess the association of NQO1 and tumor necrosis factor (TNF) polymorphisms with PD. A meta-analysis was performed that included data from 15 studies comprising 2,858 patients and 2,907 healthy controls. The results showed that TNF-1031 (rs1799964) was significantly associated with PD in the recessive [P=0.0005; odds ratio (OR), 3.19; 95% confidence interval (CI), 1.66-6.13] and additive models (P=0.0006; OR, 3.15; 95% CI, 1.63-3.51). However, there was no significant association in NQO1 C609T (rs1800566) and TNF-308 (rs1800629) with PD. To the best of our knowledge, the present study is the first meta-analysis of NQO1 and TNF polymorphisms with PD demonstrating that TNF-1031 polymorphism may be a risk factor for PD under either the recessive or additive models. However, the meta-analyses did not support the involvement of NQO1 C609T and TNF-308 in the risk of PD.

Age-Dependent Effect of Every-Other-Day Feeding and Aerobic Exercise in Ubiquinone Levels and Related Antioxidant Activities in Mice Muscle

Aging affects many biochemical, cellular, and physiological processes in the organisms. Accumulation of damage based on oxidized macromolecules is found in many age-associated diseases. Coenzyme Q (Q) is one of the main molecules involved in metabolic and antioxidant activities in cells. Q-dependent antioxidant activities are importantly involved on the protection of cell membranes against oxidation. Many studies indicate that Q decay in most of the organs during aging. In our study, no changes in Q levels were found in old animals in comparison with young animals. On the other hand, the interventions, caloric restriction based on every-other-day feeding procedure, and physical exercise were able to increase Q levels in muscle, but only in old and not in young animals. Probably, this effect prevented the increase in lipid peroxidation found in aged animals and also protein carbonylation. Further, Q-dependent antioxidant activities such as NADH-cytochrome b5 reductase and NAD(P)H-quinone oxidoreductase 1 are also modulated by both exercise and every other day feeding. Taken together, we demonstrate that exercise and dietary restriction as every-other-day procedure can regulate endogenous synthesized Q levels and Q-dependent antioxidant activities in muscle, preventing oxidative damage in aged muscle.

Suppression of NAD(P)H-quinone oxidoreductase 1 enhanced the susceptibility of cholangiocarcinoma cells to chemotherapeutic agents

BackgroundCholangiocarcinoma (CCA) is highly resistant to most of the known chemotherapeutic treatments. NAD(P)H-quinone oxidoreductase 1 (NQO1) is an antioxidant/detoxifying enzyme recently recognized as an important contributor to chemoresistance in some human cancers. However, the contribution of NQO1 to chemotherapy resistance in CCA is unknown.MethodsTwo CCA cell lines, KKU-100 and KKU-M214, with high and low NQO1 expression levels, respectively, were used to evaluate the sensitivity to chemotherapeutic agents; 5-fluorouracil (5-FU), doxorubicin (Doxo), and gemcitabine (Gem). NQO1 and/or p53 expression in KKU-100 cells were knocked down by siRNA. NQO1 was over-expressed in KKU-M214 cells by transfection with pCMV6-XL5-NQO1 expression vector. CCA cells with modulated NQO1 and/or p53 expression were treated with chemotherapeutic agents, and the cytotoxicity was assessed by SRB assay. The mechanism of enhanced chemosensitivity was evaluated by Western blot analysis.ResultsWhen NQO1 was knocked down, KKU-100 cells became more susceptible to all chemotherapeutic agents. Conversely, with over-expression of NQO1 made KKU-M214 cells more resistant to chemotherapeutic agents. Western blot analysis suggested that enhanced chemosensitivity was probably due to the activation of p53-mediated cell death. Enhanced susceptibility to chemotherapeutic agents by NQO1 silencing was abolished by knockdown of p53.ConclusionsThese results suggest that inhibition of NQO1 could enhance the susceptibility of CCA to an array of chemotherapeutic agents.

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 30–50ms 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.

Depleted energy charge and increased pulmonary endothelial permeability induced by mitochondrial complex I inhibition are mitigated by coenzyme Q1 in the isolated perfused rat lung

Mitochondrial dysfunction is associated with various forms of lung injury and disease that also involve alterations in pulmonary endothelial permeability, but the relationship, if any, between the two is not well understood. This question was addressed by perfusing isolated intact rat lung with a buffered physiological saline solution in the absence or presence of the mitochondrial complex I inhibitor rotenone (20μM). Compared to control, rotenone depressed whole lung tissue ATP from 5.66±0.46 (SEM) to 2.34±0.15µmol·g−1 dry lung, with concomitant increases in the ADP:ATP and AMP:ATP ratios. Rotenone also increased lung perfusate lactate (from 12.36±1.64 to 38.62±3.14µmol·15min−1 perfusion·g−1 dry lung) and the lactate:pyruvate ratio, but had no detectable impact on lung tissue GSH:GSSG redox status. The amphipathic quinone coenzyme Q1 (CoQ1; 50μM) mitigated the impact of rotenone on the adenine nucleotide balance, wherein mitigation was blocked by NAD(P)H-quinone oxidoreductase 1 or mitochondrial complex III inhibitors. In separate studies, rotenone increased the pulmonary vascular endothelial filtration coefficient (Kf) from 0.043±0.010 to 0.156±0.037ml·min−1·cm H2O−1·g−1 dry lung, and CoQ1 protected against the effect of rotenone on Kf. A second complex I inhibitor, piericidin A, qualitatively reproduced the impact of rotenone on Kf and the lactate:pyruvate ratio. Taken together, the observations imply that pulmonary endothelial barrier integrity depends on mitochondrial bioenergetics as reflected in lung tissue ATP levels and that compensatory activation of whole lung glycolysis cannot protect against pulmonary endothelial hyperpermeability in response to mitochondrial blockade. The study further suggests that low-molecular-weight amphipathic quinones may have therapeutic utility in protecting lung barrier function in mitochondrial insufficiency.

Identification of Imperative Enzymes by Differential Protein Expression in Picrorhiza kurroa Under Metabolite Accumulating and Non-accumulating Conditions

Picrorhiza kurroa, an endangered medicinal plant found in the North-Western Himalayan region has a number of medicinal properties due to the presence of metabolites picroside-I and picroside-II. It is used in various herbal formulations like Picroliv, Livokin, Picrolax, Livomap, Tefroliv etc. Review of literature revealed that no information is available as of today on the proteome analysis of Picrorhiza kurroa. Hence, we aim to analyse the difference in proteome of Picrorhiza kurroa in response to ~ 17 times higher content of picroside-I at 15°C as compared to its content at 25°C. Thus, differential protein expression was studied. Densitometry analysis of SDS-PAGE gels of samples under two differential conditions of temperature revealed the presence of distinct set of proteins under picroside-I accumulating (15°C) versus non-accumulating (25°C) conditions. Mass spectrometric analysis of these proteins using MALDI-TOF MS followed by protein identification using database search on MASCOT search engine gave interesting results. The significant proteins identified were NAD(P)H-quinone oxidoreductase subunit K, shikimate kinase, ribulose bisphosphate carboxylase small chain and fructokinase fragment. Hence, these findings throw light on the involvement of these enzymes in the crucial physiological processes of Picrorhiza kurroa and can provide an insight into the biosynthesis of picrosides and other secondary metabolites.

Differential regulation of detoxification enzymes in hepatic and mammary tissue by hops (Humulus lupulus) in vitro and in vivo.

Hops contain the phytoestrogen, 8-prenylnaringenin, and the cytoprotective compound, xanthohumol (XH). XH induces the detoxification enzyme, NAD(P)H-quinone oxidoreductase (NQO1) in vitro; however, the tissue distribution of XH and 8-prenylnaringenin and their tissue-specific activity have not been analyzed. An orally administered hop extract and subcutaneously injected XH were administered to Sprague-Dawley rats over 4 days. LC-MS-MS analysis of plasma, liver, and mammary gland revealed that XH accumulated in liver and mammary glands. Compared with the low level in the original extract, 8-prenylnaringenin was enriched in the tissues. Hops and XH-induced NQO1 in the liver, while only hops reduced NQO1 activity in the mammary gland. Mechanistic studies revealed that hops modulated NQO1 through three mechanisms. In liver cells, (i) XH modified Kelch-like ECH-associated protein leading to nuclear factor (erythroid-derived 2)-like 2 (Nrf2) translocation and antioxidant response element (ARE) activation; (ii) hop-mediated ARE induction was partially mediated through phosphorylation of Nrf2 by PKC; (iii) in breast cells, 8-prenylnaringenin reduced NQO1 likely through binding to estrogen receptorα, recruiting Nrf2, and downregulating ARE-regulated genes. XH and 8-prenylnaringenin in dietary hops are bioavailable to the target tissues. While hops and XH might be cytoprotective in the liver, 8-prenylnaringenin seems responsible for hop-mediated NQO1 reduction in the mammary gland.

Sulforaphane protects hearts from early injury after experimental transplantation

Oxidative stress is a major factor in the development of ischemia reperfusion injury (IRI) after heart transplantation. The isothiocyanate found in broccoli - sulforaphane (SFN) - is an indirect antioxidant that acts by inducing Nrf2-dependent Phase 2 enzymes, such as NAD(P)H-quinone oxidoreductase 1, glutathione reductase, glutathione peroxidase, and the cardioprotective enzymes, haemoxygenase-1 (HO-1) and thioredoxin (Trx-1), participate in adaptive and protective responses to oxidative stress and various inflammatory stimuli. The aim of this study was to ameliorate IRI following heart transplants by recipient preconditioning. Male Lewis rats were randomly divided into groups of n=10 animals each for heart donation. Heart grafts underwent 18 hours of cold storage in histidine-tryptophanketoglutarate (HTK) solution. Preconditioning of recipients with sulforaphane was performed 24 hours before transplantation. SFN significantly decreased serum levels of TnT, CK, CK-MB, LDH, AST, and ALT, which correlated with better graft function scores and improved survival prognosis. Pretreated recipients showed significantly decreased expression of iNOS, caspase 3, and HIF-1a. Our results indicate that recipient preconditioning with SFN protects the heart from IRI after experimental transplantation.

Abstract 572: Induction of NAD(P)H-Quinone oxidoreductase 1 by antioxidants in female ACI rats is associated with decrease in oxidative DNA damage and inhibition of estrogen-induced breast cancer

Abstract Exact mechanisms underlying the initiation and progression of estrogen-related cancers are not clear. Literature evidence and our studies strongly support the role of estrogen metabolism-mediated oxidative stress in estrogen-induced breast carcinogenesis. We have recently demonstrated that antioxidants vitamin C and butylated hydroxyanisole (BHA) or estrogen metabolism inhibitor α-naphthoflavone (ANF) inhibit 17β-estradiol (E2)-induced mammary tumorigenesis in female ACI rats. The objective of the current study was to identify the mechanism of antioxidant-mediated protection against E2-induced DNA damage and mammary tumorigenesis. Female ACI rats were treated with E2 in the presence or absence of vitamin C or BHA or ANF for up to 240 days. Nuclear factor erythroid 2-related factor 2 (NRF2) and NAD(P)H-Quinone oxidoreductase 1 (NQO1) mRNA and protein levels were suppressed in E2-exposed mammary tissue and in mammary tumors after treatment of rats with E2 for 240 days. This suppression was overcome by co-treatment of rats with E2 and vitamin C or BHA. Vitamin C and BHA significantly increased NQO1 levels after 120 days. 8-Hydroxydeoxyguanosine (8-OHdG) levels were higher in E2-exposed mammary tissue and in mammary tumors compared to age-matched controls. Vitamin C or BHA treatment significantly decreased E2-mediated increase in 8-OHdG levels in the mammary tissue. In vitro studies using silencer RNA confirmed the role of NQO1 in prevention of oxidative DNA damage. Our studies further demonstrate that NQO1 upregulation by antioxidants is mediated through NRF2. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 572. doi:1538-7445.AM2012-572