Chlorella ethanol extract induced phase II enzyme through NFE2L2 (nuclear factor [erythroid-derived] 2-like 2, NRF2) activation and protected ethanol-induced hepatoxicity.

In this study, we investigated the hepatoprotective effects of ethanol extracts from Chlorella vulgaris (CH) on animals. We measured its effect on the quinone reductase (QR) activity in Hepa1c1c7 cells, finding that CH induced a significantly higher QR activity in these cells. We isolated the active fraction (CH F4-2) from CH using chromatography methods. CH F4-2 may activate cellular antioxidant enzymes through upregulation of the Nrf2 pathway in hepatocarcinoma cells with CH F4-2 (25.0-200 µg/mL) for 48 h. Furthermore, CH F4-2 increased the expression of NQO1 [ NAD(P)H: quinone oxidoreductase, also known as QR], heme oxygenase-1, and glutathione-S-transferase P. Moreover, we found that ethanol-induced hepatic pathological changes-elevations in glutamic oxaloacetic transaminase, glutamic pyruvic transaminase, γ-glutamyltransferase, and lactate dehydrogenase-were significantly decreased. The inhibitory effect of CH on alcohol-induced liver injury was associated with the suppression of alcohol-induced increases in intestinal permeability. The ethanol extract from CH was found to induce QR activation, making it a potentially good candidate for a hepatoprotection agent.

Penicillipyrones A and B, meroterpenoids from a marine-derived Penicillium sp. fungus.

Penicillipyrones A (1) and B (2), two novel meroterpenoids, were isolated from the marine-derived fungus Penicillium sp. On the basis of the results of combined spectroscopic analyses, these compounds were structurally elucidated to be sesquiterpene γ-pyrones from a new skeletal class derived from a unique linkage pattern between the drimane sesquiterpene and pyrone moieties. Compound 2 elicited significant induction of quinone reductase.

A secondary mode of action of polymyxins against Gram-negative bacteria involves the inhibition of NADH-quinone oxidoreductase activity.

Polymyxin B and colistin were examined for their ability to inhibit the type II NADH-quinone oxidoreductases (NDH-2) of three species of Gram-negative bacteria. Polymyxin B and colistin inhibited the NDH-2 activity in preparations from all of the isolates in a concentration-dependent manner. The mechanism of NDH-2 inhibition by polymyxin B was investigated in detail with Escherichia coli inner membrane preparations and conformed to a mixed inhibition model with respect to ubiquinone-1 and a non-competitive inhibition model with respect to NADH. These suggest that the inhibition of vital respiratory enzymes in the bacterial inner membrane represents one of the secondary modes of action for polymyxins.

The antidote effect of quinone oxidoreductase 2 inhibitor against paraquat-induced toxicity in vitro and in vivo.

BACKGROUND AND PURPOSE The mechanisms of paraquat (PQ)-induced toxicity are poorly understood and PQ poisoning is often fatal due to a lack of effective antidotes. In this study we report the effects of N-[2-(2-methoxy-6H-dipyrido{2,3-a:3,2-e}pyrrolizin-11-yl)ethyl]-2-furamide (NMDPEF), a melatonin-related inhibitor of quinone oxidoreductase2 (QR2) on the toxicity of PQ in vitro & in vivo. EXPERIMENTAL APPROACH Prevention of PQ-induced toxicity was tested in different cells, including primary pneumocytes and astroglial U373 cells. Cell death and reactive oxygen species (ROS) were analysed by flow cytometry and fluorescent probes. QR2 silencing was achieved by lentiviral shRNAs. PQ (30 mg·kg(-1)) and NMDPEF were administered i.p. to Wistar rats and animals were monitored for 28 days. PQ toxicity in the substantia nigra (SN) was tested by a localized microinfusion and electrocorticography. QR2 activity was measured by fluorimetry of N-benzyldihydronicotinamide oxidation. KEY RESULTS NMDPEF potently antagonized non-apoptotic PQ-induced cell death, ROS generation and inhibited cellular QR2 activity. In contrast, the cytoprotective effect of melatonin and apocynin was limited and transient compared with NMDPEF. Silencing of QR2 attenuated PQ-induced cell death and reduced the efficacy of NMDPEF. Significantly, NMDPEF (4.5 mg·kg(-1)) potently antagonized PQ-induced systemic toxicity and animal mortality. Microinfusion of NMDPEF into SN prevented severe behavioural and electrocortical effects of PQ which correlated with inhibition of malondialdehyde accumulation in cells and tissues. CONCLUSIONS AND IMPLICATIONS NMDPEF protected against PQ-induced toxicity in vitro and in vivo, suggesting a key role for QR2 in the regulation of oxidative stress.

An unexpected effect of 5-MCA-NAT in chick retinal development.

Luzindole is an unselective antagonist of the melatonin receptors and melatonin's other binding sites, although some exceptions have been observed in chick retinal neurodevelopment, where this unselective antagonist does not block melatonin's inhibitory effect on the adenylate cyclase enzyme, probably due to the presence of some other melatonin receptor(s) or binding site(s). The present study investigated the modulation of cyclic adenosine 3'-5'-monophosphate (cAMP) levels via MT3 melatonin-binding sites, located within the QR2 (dihydronicotinamide riboside: quinone oxidoreductase 2) enzyme, by observing the response to luzindole. Embryonic and post-hatch retinas, incubated with a selective agonist for the MT3 melatonin-binding site 5-methoxycarbonylamino-N-acetyltryptamine (5-MCA-NAT, 10 or 100 nM), had an increase in cAMP accumulation relative to control retinas. Luzindole (5microM) inhibited the 5-MCA-NAT stimulatory effect at all ages tested. The agonist 5-MCA-NAT enhanced the melatonin inhibitory effect on cAMP levels stimulated by forskolin (5microM), but not the stimulatory forskolin effect. The results suggest that MT3 melatonin-binding sites are present in embryonic and post-hatch chick retinas and that luzindole more selectively blocks the 5-MCA-NAT effect on cAMP accumulation than it blocks the melatonin inhibitory effect via G protein-coupled receptors in chick retinal neurodevelopment.

Evaluation of isothiocyanates as potent inducers of carcinogen-detoxifying enzymes in the urinary bladder: critical nature of in vivo bioassay.

Deficiency of carcinogen-detoxifying phase 2 enzymes, such as glutathione S-transferase (GST) and NAD(P)H:quinone oxidoreductase 1 (NQO1), increases bladder cancer risk in humans. We report that several isothiocyanates (ITCs) that have not been previously examined, 1-methylbutyl ITC in particular, potently and preferentially induce both GST and NQO1 in the rat bladder. Comparison of 25 ITCs that are closely related in chemical structures showed that a 3-5-carbon aliphatic side chain with a methyl group attached to the alpha carbon was crucial for maximal inducer activity in the bladder. Surprisingly, cell-based bioassays failed to predict the phase 2 enzyme-inducing activity of the ITCs in the bladder. Furthermore, although ITCs are principally metabolized in vivo to dithiocarbamates (DTCs), which are believed to serve as the carriers of ITCs and are rapidly eliminated and concentrated in the urine, the total urinary levels of ITC plus DTC did not correlate with the degree of GST and NQO1 induction by the ITCs in the bladder of rats. Thus, several underappreciated ITCs are exceedingly potent inducers of GST and NQO1 in the rat bladder but were predicted neither by in vitro bioassays of phase 2 enzyme induction nor by their appearance or concentration in urine in vivo.

New ligands at the melatonin binding site MT(3).

The third melatonin binding site, MT3 is a non-classical one since it is not a seven transmembrane domains receptor, but an enzyme, quinone reductase 2. A major concern for the study of the physiological role of this site is the lack of specific ligands, permitting to more accurately dissect the pathways linked to the activation of MT3. Indeed, in the course of finding new ligands, we identified a new series of compounds with affinity to the binding site in the nM range, particularly 2,3-dimethoxy 7-hydroxy 10-methyl 5H 10H indeno(1,2-b)indol-10-one (DMHMIO), with a Ki of 190 pM. Based on slightly different and novel synthons compared to most of the compounds used in melatonin pharmacology studies, these compounds offer new perspective for the description of the melatonin pathways, so much more by not having any affinity towards the MT1 and MT2 'classical' melatonin receptors.

A unique cytosolic activity related but distinct from NQO1 catalyses metabolic activation of mitomycin C.

Mitomycin C (MMC) is a prototype bioreductive drug employed to treat a variety of cancers including head and neck cancer. Among the various enzymes, dicoumarol inhibitable cytosolic NAD(P)H:quinone oxidoreductase1 (NQO1) was shown to catalyse bioreductive activation of MMC leading to cross-linking of the DNA and cytotoxicity. However, the role of NQO1 in metabolic activation of MMC has been disputed. In this report, we present cellular and animal models to demonstrate that NQO1 may play only a minor role in metabolic activation of MMC. We further demonstrate that bioreductive activation of MMC is catalysed by a unique cytosolic activity which is related but distinct from NQO1. Chinese hamster ovary (CHO) cells were developed that permanently express higher levels of cDNA-derived NQO1. These cells showed significantly increased protection against menadione toxicity. However, they failed to demonstrate higher cytotoxicity due to exposure to MMC under oxygen (normal air) or hypoxia, as compared to the wild-type control CHO cells. Disruption of the NQO1 gene by homologous recombination generated NQO1-/- mice that do not express the NQO1 gene resulting in the loss of NQO1 protein and activity. The cytosolic fractions from liver and colon tissues of NQO1-/- mice showed similar amounts of DNA cross-linking upon exposure to MMC, as observed in NQO1+/+ mice. The unique cytosolic activity that activated MMC in cytosolic fractions of liver and colon tissues of NQO1-/- mice was designated as cytosolic MMC reductase. This activity, like NQO1, was inhibited by dicoumarol and immunologically related to NQO1.

Rosemary extract and carnosol stimulate rat liver glutathione-S-transferase and quinone reductase activities.

The effects of dietary intake and intraperitoneal (i.p.) administration of an extract of the spice rosemary and of the rosemary constituent carnosol on the liver activities of glutathione-S-transferase (GST) and NAD(P)H-quinone reductase (QR) in the female rat were evaluated. Rosemary extract at concentrations from 0.25 to 1.0% (by wt.) in the diet resulted in a significant 3.5- to 4.5-fold increase in liver GST and a 3.3- to 4.0-fold increase in liver QR activities compared to controls. Carnosol supplemented in the diet at levels from 0.01 to 1.0% did not enhance GST activity. When rosemary extract and carnosol were administered i.p. there was a significant increase in liver GST and QR activities. The injection of rosemary extract (200 mg/kg) was associated with 1.5-fold and 3.2-fold increases in GST and QR activities, respectively, compared to controls. The injection of carnosol at doses from 100 to 400 mg/kg was associated with 1.6- to 1.9-fold increases in GST activity and 3.1- to 4.8-fold increases in QR activity, compared to controls. These data indicate that rosemary extract in the diet or injected i.p. and carnosol administered i.p. are effective enhancers of the in vivo activity of liver GST and QR in the female rat.

Expression and characterization of the 66-kilodalton (NQO3) iron-sulfur subunit of the proton-translocating NADH-quinone oxidoreductase of Paracoccus denitrificans.

The proton-translocating NADH-quinone oxidoreductase (NDH-1) of Paracoccus denitrificans is composed of at least 14 dissimilar subunits which are designated NQO1-14 and contains one noncovalently bound FMN and at least five EPR-visible iron-sulfur clusters (N1a, N1b, N2, N3, and N4) as prosthetic groups. Comparison of the deduced primary structures of the subunits with consensus sequences for the cofactor binding sites has predicted that NQO1, NQO2, NQO3, NQO9, and probably NQO6 subunits are cofactor binding subunits. Previously, we have reported that the NQO2 (25 kDa) subunit was overexpressed as a water-soluble protein in Escherichia coli and was found to ligate a single [2Fe-2S] cluster with rhombic symmetry (gx,y,z = 1.92, 1.95, and 2.00) (Yano, T., Sled', V.D., Ohnishi, T., and Yagi, T. (1994) Biochemistry 33, 494-499). In the present study, the NQO3 (66 kDa) subunit, which is equivalent to the 75-kDa subunit of bovine heart Complex I, was overexpressed in E. coli. The expressed NQO3 subunit was found predominantly in the cytoplasmic phase and was purified by ammonium sulfate fractionation and anion-exchange chromatography. The chemical analyses and UV-visible and EPR spectroscopic studies showed that the expressed NQO3 subunit contains at least two distinct iron-sulfur clusters: a [2Fe-2S] cluster with axial EPR signals (g perpendicular, parallel = 1.934 and 2.026, and L perpendicular parallel = 1.8 and 3.0 millitesla) and a [4Fe-4S] cluster with rhombic symmetry (gx,y,z = 1.892, 1.928, and 2.063, and Lx,y,z = 2.40, 1.55, and 1.75 millitesla). The midpoint redox potentials of [2Fe-2S] and [4Fe-4S] clusters at pH 8.6 are -472 and -391 mV, respectively. The tetranuclear cluster in the isolated NQO3 subunit is sensitive toward oxidants and converts into [3Fe-4S] form. The assignment of these iron-sulfur clusters to those identified in the P. denitrificans NDH-1 enzyme complex and the possible functional role of the NQO3 subunit is discussed.

Induction of quinone reductase and glutathione in bone marrow cells by 1,2-dithiole-3-thione: effect on hydroquinone-induced cytotoxicity.

Stromal cells from bone marrow are susceptible to toxicity induced by several redox-active metabolites of benzene, including hydroquinone (HQ). We have previously shown that tert-butyl-hydroquinone (tBHQ) can induce quinone reductase (QR) in bone marrow stroma as well as protect stromal cells against HQ-induced toxicity. Current studies investigate the underlining mechanisms of chemoprotection against HQ in DBA/2- and C57Bl/6-derived bone marrow stromal cells. The chemoprotector 1,2-dithiole-3-thione (DTT) has been used in these studies due to tBHQ toxicity to stromal cells at higher concentrations. Pretreatment of cells with DTT prior to HQ administration protected cells against HQ-induced toxicity. DTT induced QR activity in a dose-dependent manner in stromal cells from both strains of mice. However, there were no corresponding changes in glutathione transferase activity. DTT also increased cytosolic glutathione (GSH) concentrations by approximately 85% in both strains. Since bone marrow stroma consists primarily of fibroblasts and macrophages, we also evaluated QR activity in the separate cell types from the two strains of mice. There were differences in basal and DTT-induced QR activity between fibroblasts and macrophage cells derived from the same strain of mice, as well as the expected differences between strains. Additionally, dicoumarol, an inhibitor of QR activity, potentiated HQ-induced toxicity in both strains of bone marrow stromal cells. Thus, cellular glutathione, QR activity, and their inducibility by chemoprotective agents such as DTT may prove to be important factors in chemically induced bone marrow toxicity and carcinogenicity.

In vitro effect of mercury on aryl hydrocarbon hydroxylase, quinone reductase, catecholamine-O-methyltransferase and glucose-6-phosphate dehydrogenase activities in term human placenta.

The effect of HgCl2 on human term placental aryl hydrocarbon hydroxylase (AHH), quinone reductase (QR), catecholamine-O-methyltransferase (COMT), and glucose-6-phosphate dehydrogenase (G-6-PD) enzyme activities was studied after incubation of placental explants with the salt for either a 6 or 24 hr period. Mercury (Hg) increased the activities of AHH, QR and COMT, but decreased that of G-6-PD. The increases in enzyme activities, as well as the decrease in G-6-PD activity observed were in all cases time- and dose-dependent. The data suggest that Hg exerts an enhancing effect on the activity of placental phase I enzyme (AHH) and phase II enzymes (QR and COMT). This enhancement may be due to increased de novo synthesis, elimination of some suppressing agent(s), or the decreased breakdown of enzyme protein. Also, the inhibitory effect of Hg on G-6-PD activity appears to indicate that this enzyme is appreciably more sensitive to Hg than the other three enzymes. These findings may imply increased cellular resistance to Hg toxicity. The altered state of activity may also be used as a tool for monitoring exposure to this metal.