Aqueous and Ethanol Extracts of Daylily Flower (Hemerocallis fulva L.) Protect HUVE Cells Against High Glucose.

The content of several phenolic acids and flavonoids in aqueous extract (AE) and ethanol extract (EE) of daylily flower (Hemerocallis fulva L.) was analyzed. The effects of AE or EE at 0.5%, 1%, or 2% in HUVE cells against high glucose-induced cell death, oxidative, and inflammatory damage were examined. Results showed that seven phenolic acids and seven flavonoids could be detected in AE or EE, in the range of 29 to 205 and 41 to 273 mg/100 g, respectively. Compared with the control groups, high glucose raised the activity of caspase-3 and caspase-8; suppressed Bcl-2 mRNA expression and increased Bax mRNA expression; and induced HUVE cells apoptosis. The pretreatments from AE or EE at 1% or 2% reduced caspase-3 activity and Bax mRNA expression, and enhanced cell viability. High glucose decreased glutathione content; stimulated the production of reactive oxygen species, interleukin-6, tumor necrosis factor-alpha, and prostaglandin E2 ; raised the activity of cyclooxygenase-2 and nuclear factor kappa B p50/65 binding; and reduced the activity of glutathione peroxidase, glutathione reductase, and catalase in HUVE cells. AE pretreatments at 1% and 2% reversed these changes. These novel findings suggested that daylily flower was rich in phytochemicals, and could be viewed as a potent functional food against diabetes.

Alleviation of cadmium-induced oxidative stress by trehalose via inhibiting the Nrf2-Keap1 signaling pathway in primary rat proximal tubular cells.

Nuclear factor erythroid 2-related factor 2 (Nrf2) is a transcription factor that regulates a cluster of oxidative stress-inducible genes in cells. Here, we aimed to investigate whether trehalose (Tre) protects primary rat proximal tubular (rPT) cells against cadmium (Cd)-induced oxidative stress via Nrf2 antioxidant pathway. Data showed that Tre treatment inhibited Nrf2 nuclear translocation and restored the decline in Kelch-like ECH-associated protein 1 (Keap1) protein level in Cd-exposed rPT cells. Moreover, Cd-activated Nrf2 target genes, including phase II detoxifying enzymes, that is, NAD(P)H quinone oxidoreductase 1 and heme oxygenase-1, direct antioxidant proteins, that is, glutathione peroxidase, superoxide dismutase, catalase, and glutathione biosynthesis-related proteins, that is, glutamatecysteine ligase catalytic subunit, glutamate cysteine ligase modifier subunit, and glutathione reductase, were all downregulated by co-treatment with Tre. Collectively, these findings demonstrate that Tre treatment alleviates Cd-induced oxidative stress in rPT cells by inhibiting the Nrf2-Keap1 signaling pathway.

Unexpected Thiols Triggering Photoluminescent Enhancement of Cytidine Stabilized Au Nanoclusters for Sensitive Assays of Glutathione Reductase and Its Inhibitors Screening.

The photoluminescence (PL) of nonthiolate ligand capped Au nanoclusters (NCs) is usually quenched by thiols due to the tight adsorption of thiols to the Au surface and formation of larger non-PL species. However, we here report an unexpected PL enhancement of cytidine stabilized Au (AuCyt) NCs triggered by thiols, such as reduced glutathione (GSH) at sub-µM level, while such phenomena have not been observed for Au NCs capped with similar adenosine/cytidine nucleotides. The mass spectroscopic results indicate that this enhancement may be caused by the formation of smaller, but highly fluorescent, Au species etched by thiols. This enables the sensitive detection of GSH from 20 nM to 3 µM, with an ultralow detection limit of 2.0 nM. Moreover, the glutathione reductase (GR) activity can be determined by the initial rate of GSH production, i.e., the maximum PL increasing rate, with a linear range of 0.34-17.0 U/L (1 U means reduction of 1.0 µmol of oxidized glutathione per min at pH 7.6 at 25 °C) and a limit of detection of 0.34 U/L. This method allows the accurate assays of GR in clinical serum samples as well as the rapid screening of GR inhibitors, indicating its promising biomedical applications.

Synthesis and evaluation of 1,4-naphthoquinone ether derivatives as SmTGR inhibitors and new anti-schistosomal drugs.

Investigations regarding the chemistry and mechanism of action of 2-methyl-1,4-naphthoquinone (or menadione) derivatives revealed 3-phenoxymethyl menadiones as a novel anti-schistosomal chemical series. These newly synthesized compounds (1-7) and their difluoromethylmenadione counterparts (8, 9) were found to be potent and specific inhibitors of Schistosoma mansoni thioredoxin-glutathione reductase (SmTGR), which has been identified as a potential target for anti-schistosomal drugs. The compounds were also tested in enzymic assays using both human flavoenzymes, i.e. glutathione reductase (hGR) and selenium-dependent human thioredoxin reductase (hTrxR), to evaluate the specificity of the inhibition. Structure-activity relationships as well as physico- and electro-chemical studies showed a high potential for the 3-phenoxymethyl menadiones to inhibit SmTGR selectively compared to hGR and hTrxR enzymes, in particular those bearing an α-fluorophenol methyl ether moiety, which improves anti-schistosomal action. Furthermore, the (substituted phenoxy)methyl menadione derivative (7) displayed time-dependent SmTGR inactivation, correlating with unproductive NADPH-dependent redox cycling of SmTGR, and potent anti-schistosomal action in worms cultured ex vivo. In contrast, the difluoromethylmenadione analog 9, which inactivates SmTGR through an irreversible non-consuming NADPH-dependent process, has little killing effect in worms cultured ex vivo. Despite ex vivo activity, none of the compounds tested was active in vivo, suggesting that the limited bioavailability may compromise compound activity. Therefore, future studies will be directed toward improving pharmacokinetic properties and bioavailability.

Targeting the intersubunit cavity of Plasmodium falciparum glutathione reductase by a novel natural inhibitor: computational and experimental evidence.

Glutathione reductase (GR), a homodimeric FAD-dependent disulfide reductase, is essential for redox homeostasis of the malaria parasite Plasmodium falciparum and has been proposed as an antimalarial drug target. In this study we performed a virtual screening against PfGR, using the structures of about 170,000 natural compounds. Analysis of the two top-scoring molecules, TTB and EPB, indicated that these ligands are likely to interact with the homodimer intersubunit cavity of PfGR with high binding energy scores of -9.67 and -9.60kcal/mol, respectively. Both compounds had a lower affinity for human GR due to differences in structure and electrostatic properties. In order to assess the putative interactions in motion, molecular dynamics simulations were carried out for 30ns, resulting in TTB being more dynamically and structurally favored than EPB. A closely related compound MDPI 21618 was tested on recombinant PfGR and hGR, resulting in IC50 values of 11.3±2.5µM and 10.2±1.7µM, respectively. Kinetic characterization of MDPI 21618 on PfGR revealed a mixed-type inhibition with respect to glutathione disulfide (Ki=9.7±2.3µM) and an uncompetitive inhibition with respect to NADPH. Furthermore, MDPI 21618 was found to inhibit the growth of the chloroquine-sensitive P. falciparum strain 3D7 with an IC50 of 3.2±1.9µM and the chloroquine-resistant Dd2 strain with an IC50 of 3.2+1.6µM. In drug combination assays with chloroquine, artemisinin, or mefloquine MDPI 21618 showed an antagonistic action, which might suggest partially overlapping routes of action. This study further substantiates research on PfGR as a potential antimalarial drug target.

Determination of antiplasmodial activity and binding affinity of selected natural products towards PfTrxR and PfGR.

In our study, the binding affinities of selected natural products towards PfTrxR, PfGR, human TrxR and human GR were determined using a mass spectrometry based ligand binding assay. The in vitro antimalarial activity and cytotoxicity of these ligands were also determined. Catharanthine, 11-(OH)-coronaridine, hernagine, vobasine and hispolone displayed antiplasmodial activity against PfK1 (IC50 = 0.996-3.63 microg/mL).

Anticancer therapeutics that target selenoenzymes: synthesis, characterization, in vitro cytotoxicity, and thioredoxin reductase inhibition of a series of gold(I) complexes containing hydrophilic phosphine ligands.

Gold(I) complexes bearing water-soluble phosphine ligands, including 1,3,5-triaza-7-phosphaadamantane (PTA), 3,7-diacetyl-1,3,7-triaza-5-phosphabicyclo[3.3.1]nonane (DAPTA), and sodium triphenylphosphine trisulfonate (TPPTS), in combination with thionate ligands, were screened for their antiproliferative activities against human ovarian cancer cell lines A2780 either sensitive or resistant to cisplatin. In addition, the compounds were screened for their inhibition of mammalian thioredoxin reductases (TrxR), enzymes that are overexpressed in many tumor cells and contribute to drug resistance. The gold(I)-phosphine complexes efficiently inhibited cytosolic and mitochondrial TrxRs at concentrations that did not affect the related oxidoreductase glutathione reductase (GR). Additional complementary information on the enzyme metallation process and potential gold binding sites was obtained through the application of a specific biochemical assay using a thiol-tagging reagent, BIAM (biotin-conjugated iodoacetamide).

The aza-analogues of 1,4-naphthoquinones are potent substrates and inhibitors of plasmodial thioredoxin and glutathione reductases and of human erythrocyte glutathione reductase.

Various aza-analogues of 1,4-naphthoquinone and menadione were prepared and tested as inhibitors and substrates of the plasmodial thioredoxin and glutathione reductases as well as the human glutathione reductase. The replacement of one to two carbons at the phenyl ring of the 1,4-naphthoquinone core by one to two nitrogen atoms led to an increased oxidant character of the molecules in accordance with both the redox potential values and the substrate efficiencies. Compared to the 1,4-naphthoquinone and menadione, the quinoline-5,8-dione 1 and both quinoxaline-5,8-diones 5 and 6 behaved as the most efficient subversive substrates of the three NADPH-dependent disulfide reductases tested. Modulation of these parameters was observed by alkylation of the aza-naphthoquinone core.

Effects of nicotine and vitamin E on glutathione reductase activity in some rat tissues in vivo and in vitro.

Effects of nicotine, and nicotine+vitamin E on glutathione reductase (Glutathione: NADP(+) oxidoreductase, EC 1.8.1.7) activity in the muscle, heart, lungs, testicles, kidney, stomach, brain and liver tissues were investigated in vivo and also in vitro. The groups were: nicotine [0.5 mg/kg/day, intraperitoneal (i.p.)]; nicotine+vitamin E [75 mg/kg/day, intragastric (i.g.)]; and control group (receiving only vehicles). There were eight rats per group and supplementation period was 3 weeks. The results showed that nicotine (0.5 mg/kg, i.p.) inhibited glutathione reductase activity significantly in the liver, lungs, heart, stomach, kidney, and testicles by approximately 61.5%, approximately 65%, approximately 70.5%, approximately 72.5%, approximately 64% and approximately 71.5%, respectively, while it had activated glutathione reductase activity in the brain by approximately 11.8%, and had no effect on the muscle glutathione reductase activity. Vitamin E supplementation prevented this nicotine-induced decrease in glutathione reductase activity in liver, lungs, heart, stomach, and kidney. However, it did not prevent this nicotine-induced decrease in testicles. In vitro studies were also carried out to elucidate the effects of nicotine and vitamin E on glutathione reductase activity. In vitro results correlated well with in vivo experimental results in liver, lungs, heart, stomach, and testicular tissues. These results show that vitamin E administration generally restores the inactivation of glutathione reductase activity due to nicotine administration in various rat tissues in vivo, and also in vitro.

Effect of tea catechins on the change of glutathione levels caused by Pb(++) in PC12 cells.

Our previous research showed that tea catechins could significantly increase the viability of lead-exposed PC12 cells. Whereas the cellular thiol status is known to be responsible for protecting against lead-induced toxicity, whether the role of tea catechins on lead-induced PC12 cell toxicity is related to the metabolism of intracellular thiol compounds remained vague. In the present study, it was found that Pb(2+) significantly decreased reduced glutathione (GSH)/oxidative glutathione (GSSG) and protein sulfhydryl groups (PSH)/glutathione-protein mixed disulfide (GSSP) ratios as well as glutathione reductase activities in a concentration-dependent manner. Both (-)-epicatechin and (-)-epicatechin gallate (ECG) supplementation resulted in an increased GSH/GSSG ratio and glutathione reductase activities. The galloylated catechins (ECG or (-)-epigallocatechin gallate) treatment significantly decreased the GSSP levels and increased the intracellular PSH/GSSP ratio in lead-exposed PC12 cells. To our surprise, as compared with the group treated by lead acetate, 100 microM EGC treatment following lead exposure significantly decreased GSH/GSSG and PSH/GSSP ratios, as well as glutathione reductase activities. The results suggested that the effect of tea catechins on the intracellular thiols status in PC12 cells was different, which may be related to their chemical structures and/or regulation of special gene expression properties.

Apoptotic and anti-adhesion effect of ajoene, a garlic derived compound, on the murine melanoma B16F10 cells: possible role of caspase-3 and the alpha(4)beta(1) integrin.

In this study we evaluated the hypothesis that the antitumor activity of ajoene could be associated with its apoptosis-inducing effect, and with its ability to block the expression of the alpha(4)beta(1) integrin, in the murine melanoma B16F10 cells. Ajoene induced a significant reduction in B16F10 viability (IC(50)=62 microM), in a dose-dependent manner. Flow cytometric analysis showed that the cytotoxic effect of this compound was associated with caspase-3 activation. Ajoene at 25 microM altered the alpha(4)beta(1) integrin expression on B16F10, and induced a significant reduction in the adhesion of these cells to an endothelial cell monolayer.

Inhibition of glutathione reductase by cadmium ion in some rabbit tissues and the protective role of dietary selenium.

This study is aimed at investigating the inhibitory effect of cadmium ion on glutathione reductase activity of rabbit brain and liver and the relationship of this effect with dietary selenium. For this purpose, one group of New Zealand rabbits were fed a selenium-deficient diet, another group was fed a selenium-rich diet, and the control group was fed a normal diet. The brain and liver tissues of these groups were investigated for the in vitro inhibitory effects of Cd2+ on glutathione reductase activity. For liver, the percentage inhibition of glutathione reductase by 40 nmol/mg protein of Cd2+ was similar for selenium-deficient and control groups, but significantly lower in the selenium-rich group. For brain tissues, there was no difference with respect to cadmium inhibition of glutathione reductase in all three groups.

Effect of the herbicide 4-CPA on human erythrocyte antioxidant enzymes in vitro.

To investigate the possible role of oxygen free radicals and oxidant stress in the toxic effects of phenoxyherbicides, we studied the in vitro effect of 4-chlorophenoxyacetic acid (4-CPA) on various human erythrocyte antioxidant enzymes, namely glucose-6-phosphate dehydrogenase, catalase, selenium-dependent glutathione peroxidase, glutathione reductase and Cu/Zn-superoxide dismutase. 4-CPA added in a dose of 1 ppm to human erythrocytes for 1 h caused a significant reduction in glucose-6-phosphate dehydrogenase (P <0.001) and catalase (P <0.001) activities, but did not significantly affect the activities of other enzymes. Such selective inactivation of specific erythrocyte antioxidant enzymes may play a role in the toxic effects of phenoxyherbicides.

Cellular titration of apoptosis with steady state concentrations of H(2)O(2): submicromolar levels of H(2)O(2) induce apoptosis through Fenton chemistry independent of the cellular thiol state.

Apoptosis was studied under conditions that mimic the steady state of H(2)O(2) in vivo. This is at variance with previous studies involving a bolus addition of H(2)O(2), a procedure that disrupts the cellular homeostasis. The results allowed us to define three phases for H(2)O(2)-induced apoptosis in Jurkat T-cells with reference to cytosolic steady state concentrations of H(2)O(2) [(H(2)O(2))(ss)]: (H(2)O(2))(ss) values below 0.7 microM elicited no effects; (H(2)O(2))(ss) approximately 0.7-3 microM induced apoptosis; and (H(2)O(2))(ss) > 3 microM yielded no additional apoptosis and a gradual shift towards necrosis as the mode of cell death were observed. H(2)O(2)-induced apoptosis was not affected by either BCNU, an inhibitor of glutathione reductase, or diamide, a compound that reacts both with low-molecular weight and protein thiols, or selenols. Glutathione depletion, accomplished by incubating cells either with buthionine sulfoximine or in cystine-free medium, rendered cells more sensitive to H(2)O(2)-induced apoptosis, but did not change the threshold and saturating concentrations of H(2)O(2) that induced apoptosis. Two unrelated metal chelators, desferrioxamine and dipyridyl, strongly protected against H(2)O(2)-induced apoptosis. It may be concluded that, under conditions of H(2)O(2) delivery that mimic in vivo situations, the oxidative event that triggers the induction of apoptosis by H(2)O(2) is a Fenton-type reaction and is independent of the thiol or selenium states of the cell.

Dose response of carboplatin-induced hearing loss in rats: antioxidant defense system.

Carboplatin, a platinum-containing anticancer drug, is currently being used against a variety of cancers. However, a single high dose of carboplatin is ototoxic in cancer patients. This is the first study to show carboplatin-induced hearing loss in a rat model. Male Wistar rats were divided into five groups and treated as follows: (1) control (saline, intraperitoneally (i.p.)); (2) carboplatin (64 mg/kg, i.p.); (3) carboplatin (128 mg/kg i.p.); (4) carboplatin (192 mg/kg, i.p.) and (5) carboplatin (256 mg/kg, i.p.). Animals in all groups were sedated with ketamine/xylazine and auditory brain-evoked responses (ABRs) were recorded before and 4 days after treatments. The animals were sacrificed on the fourth day and cochleae were harvested and analyzed. Carboplatin dose-dependently decreased body weight. However, at higher doses of carboplatin (192 and 256 mg/kg), there was a significant elevation of hearing threshold shifts at clicks, 4, 8, 16 and 32 kHz tone burst stimuli. The higher doses of carboplatin (192 and 256 mg/kg) significantly increased cochlear lipid peroxidation (132 and 146% of control) and depleted cochlear glutathione levels (66 and 63% of control), respectively. The antioxidant enzyme activities such as superoxide dismutase, catalase, glutathione peroxidase, glutathione reductase, and glutathione-S-transferase (GST) depressed significantly at higher doses of carboplatin. The data suggest that higher doses of carboplatin (above 128 mg/kg) induce hearing loss as evidenced by significant changes in ABRs, lipid peroxidation and antioxidants in the cochlea of rats.

Ajoene is an inhibitor and subversive substrate of human glutathione reductase and Trypanosoma cruzi trypanothione reductase: crystallographic, kinetic, and spectroscopic studies.

Ajoene ((E,Z)-4,5,9-trithiadodeca-1,6,11-triene 9-oxide), a garlic-derived natural compound, is a covalent inhibitor as well as a substrate of human glutathione reductase (GR) and Trypanosoma cruzi trypanothione reductase (TR). The 2.1-A resolution crystal structure of GR inhibited by (E)-ajoene revealed a mixed disulfide between the active site Cys58 and the CH2=CH-CH2-SO-CH2-CH=CH-S moiety of ajoene. The modified enzyme has a markedly increased oxidase activity when compared to free GR. GR reduces (Z)-ajoene with a kcat/Km of 6.8 x 10(3) M-1 s-1 yielding 4,5,9-trithiadodeca-1, 6,11-triene (deoxyajoene) and 4,8,9,13-tetrathiahexadeca-1,6,10, 15-tetraene as stable reaction products. The reaction leads also to the formation of single-electron reduced products and concomitantly superoxide anion radicals as shown by coupling the reaction to the reduction of cytochrome c. The interactions between the flavoenzymes and ajoene are expected to increase the oxidative stress of the respective cell. The antiparasitic and cytostatic actions of ajoene may at least in part be due to the multiple effects on key enzymes of the antioxidant thiol metabolism.

Suppression of inflammatory arthritis by simultaneous inhibition of nitric oxide synthase and NADPH oxidase.

TH1-type proinflammatory cytokines induce the expression of phagocytic nitric oxide synthase (NOS) and prime the membrane-bound NADPH oxidase of neutrophils and monocytes of mice so as to attain an activated state, which upon a second stimulus releases up to 6-fold increased levels of reactive oxygen species (ROS) than do unprimed phagocytes. Enhanced levels of ROS and NO deregulate inflammatory signal transduction pathways, which play a crucial role in the pathogenesis of arthritis. The antiarthritic reactivity of diphenylene iodoniumchloride (DPI), an irreversible inhibitor of NADPH oxidase and NOS, was tested in male DBA/1xB10A(4R) hybrid mice suffering from potassium peroxochromate-induced arthritis. Daily doses of 2.8 mu mol/kg of DPI sufficed to inhibit the arthritis by 50%. A complete inhibition was obtained with 10 mu mol/kg of DPI. The reduction of overt arthritic symptoms correlated well with both the reduced levels of ROS and NO in plasma of DPI-treated mice. Our data support the hypothesis that oxidative stress and nitric oxides play a pivotal role in the pathology of arthritis, which can be therapeutically targetted by NADPH oxidase- and NO synthase-inhibitors.

Antimalarial action of flavin analogues seems not be due to inhibition of glutathione reductase of host erythrocytes.

A series of 10-(4'-chlorophenyl)-3-substituted flavins (1a-f) were examined with respect to their antimalarial properties. They were tested against Plasmodium falciparum in vitro and Plasmodium vinckei vinckei in vivo. The proposition that they might act through glutathione reductase (GR) (EC 1.6.4.2) inhibition has been studied. Inhibition of P. falciparum in vitro by these compounds shows only slight variation between analogues; in contrast, inhibition of human erythrocyte GR by members of the same series is highly variable, indicating that this is probably not their primary mode of antimalarial action. Results of the P. vinckei vinckei screen showed that 10-(4'-chlorophenyl)-3-methyl,3-ethyl and 3-propyl substituted flavins are active in vivo over the dose range screened (10-70 mg/kg).

[Chemotherapeutic characterization of new nitrosourea compounds]. / Chemotherapeutische Charakterisierung neuer Nitrosoharnstoffe.

The development of new nitrosoureas is described using selected examples. Results obtained with water-soluble analogs and with compounds linked to biomolecules as for instance amino acids, oligopeptides and steroids, are presented. The pronounced antineoplastic effect of some water-soluble analogs is paralleled by an increased rate of DNA-interstrand cross-links and by an increased suppression of hematopoietic stem cells. The suppression of bone marrow stem cells is followed by their rapid regeneration. Water-soluble nitrosoureas induce significant less inhibition of glutathione reductase as compared with established compounds. With regard to long-term toxicity and carcinogenicity water-soluble are superior to established compounds as for instance BCNU. Linking of the nitrosourea moiety to amino acids and oligopeptides led to some analogs with outstanding therapeutic ratio. Out of a group of steroid-linked nitrosoureas, CNC-L-alanine-estradiol-17-ester (CNC-ala-17-E2) is chosen to demonstrate the possibility of reducing bone marrow toxicity despite unchanged or increased therapeutic activity by attachment of the nitrosourea moiety to a steroid. Results of a comparative interspecies in vitro evaluation of CNC-ala-17-E2 in transplanted MXT mammary carcinoma of the mouse, MNU-induced autochthonous rat mammary carcinoma and primary human mammary carcinomas are presented and the question is discussed to what extent in vitro activity of such receptor agents using the tumor stem cell assay reflects their in vivo activity.