Lev Weiner

Role of Oxidants and Antioxidants in the Induction of AP-1, NF-κB, and Glutathione S-Transferase Gene Expression

Transcription factors AP-1 and NF-κB have been implicated in the inducible expression of a variety of genes in response to oxidative stress. Recently, based on the observation that butylated hydroxyanisole (BHA) and pyrrolidine dithiocarbamate (PDTC) induce AP-1 binding activity and AP-1-dependent gene expression and assuming that these compounds exert an antioxidant effect, it was claimed that AP-1 is an antioxidant-responsive factor. To determine whether AP-1 can be responsive to both oxidant and antioxidant, we examined the nature of BHA and PDTC inducing activity. Using EPR spectroscopy to detect semiquinone radicals, we demonstrate the autoxidation of BHA metabolite tert-butylhydroquinone (TBHQ) to tert-butylquinone. The kinetics of TBHQ-mediated generation of ·OH radicals were monitored in intact hepatoma HepG2 cells by EPR spin trapping technique. Exogenous catalase inhibited the rate and amount of ·OH radical formation and the induction of AP-1-mediated glutathione S-transferase (GST) Ya gene expression by BHA and TBHQ, thus indicating the intermediate formation of H2O2 in the metabolism of these chemicals. Furthermore, we show that the induction of AP-1 and NF-κB activities and GST Ya gene expression by BHA and TBHQ is due to a pro-oxidant activity, since this induction was inhibited by thiol compounds N-acetyl cysteine and GSH. Similarly, induction of AP-1 and GST Ya gene expression by PDTC was inhibited by N-acetyl cysteine and GSH. The present findings do not support the notion that the induction of AP-1 by BHA, TBHQ, or PDTC is an antioxidant response and demonstrate that both AP-1 and NF-κB activities are induced by oxygen radicals.

Consecutive thermal H2 and light-induced O2 evolution from water promoted by a metal complex

Discovery of an efficient artificial catalyst for the sunlight-driven splitting of water into dioxygen and dihydrogen is a major goal of renewable energy research. We describe a solution-phase reaction scheme that leads to the stoichiometric liberation of dihydrogen and dioxygen in consecutive thermal- and light-driven steps mediated by mononuclear, well-defined ruthenium complexes. The initial reaction of water at 25°C with a dearomatized ruthenium (II) [Ru(II)] pincer complex yields a monomeric aromatic Ru(II) hydrido-hydroxo complex that, on further reaction with water at 100°C, releases H2 and forms a cis dihydroxo complex. Irradiation of this complex in the 320-to-420–nanometer range liberates oxygen and regenerates the starting hydrido-hydroxo Ru(II) complex, probably by elimination of hydrogen peroxide, which rapidly disproportionates. Isotopic labeling experiments with H217O and H218O show unequivocally that the process of oxygen–oxygen bond formation is intramolecular, establishing a previously elusive fundamental step toward dioxygen-generating homogeneous catalysis.

The mode of action of allicin: trapping of radicals and interaction with thiol containing proteins.

Allicin (thio-2-propene-1-sulfinic acid S-allyl ester) is the main biologically active component of garlic clove extracts. Its biological activity was attributed to either antioxidant activity or thiol disulfide exchange. Antioxidant properties of both allicin and its precursor, alliin (+S-allyl-L-cysteine sulfoxide), were investigated in the Fenton oxygen-radical generating system [H2O2-Fe(II)]. Using the spin trapping technique and ESR, it was found that both compounds possessed significant antioxidant activity. The reaction between allicin and L-cysteine was studied by 1H and 13C-NMR, and a S-thiolation product, S-allylmercaptocysteine, was identified. Allicin irreversibly inhibited SH-protease papain, NADP(+)-dependent alcohol dehydrogenase from Thermoanaerobium brockii (TBAD), and the NAD(+)-dependent alcohol dehydrogenase from horse liver (HLAD). All the three enzymes could be reactivated with thiol containing compounds. Papain could be reactivated with glutathione, TBAD with dithiothreitol or 2-mercaptoethanol (2-ME) but not by glutathione, while HLAD could be reactivated only with 2-ME. This study demonstrates that in addition to its antioxidant activity, the major biological effect of allicin should be attributed to its rapid reaction with thiol containing proteins.

The mode of action of allicin: its ready permeability through phospholipid membranes may contribute to its biological activity

Allicin (diallyl thiosulfinate) is the main biologically active component of the freshly crushed garlic extracts. In the present work the ability of allicin to cross through membranes (artificial and biological) was studied. Partition coefficients of allicin in water/octanol, water/hexadecane and water/phospholipids mixtures were determined. Using phospholipid vesicles loaded with hydrophilic thiols (reduced glutathione or 2-nitro-5-thiobenzoate), we observed that allicin freely permeates through phospholipid bilayers and interacts with the SH groups. The reaction rate of allicin with SH containing molecules after crossing the membrane was the same as in solution. Fast diffusion and permeation of allicin across human red blood cell membranes was also demonstrated. Allicin does not induce leakage, fusion or aggregation of membrane. The high permeability of allicin through membranes may greatly enhance the intracellular interaction with thiols.

Novel multifunctional neuroprotective iron chelator-monoamine oxidase inhibitor drugs for neurodegenerative diseases: in vitro studies on antioxidant activity, prevention of lipid peroxide formation and monoamine oxidase inhibition

Iron‐dependent oxidative stress, elevated levels of iron and of monoamine oxidase (MAO)‐B activity, and depletion of antioxidants in the brain may be major pathogenic factors in Parkinsons disease, Alzheimers disease and related neurodegenerative diseases. Accordingly, iron chelators, antioxidants and MAO‐B inhibitors have shown efficacy in a variety of cellular and animal models of CNS injury. In searching for novel antioxidant iron chelators with potential MAO‐B inhibitory activity, a series of new iron chelators has been designed, synthesized and investigated. In this study, the novel chelators were further examined for their activity as antioxidants, MAO‐B inhibitors and neuroprotective agents in vitro. Three of the selected chelators (M30, HLA20 and M32) were the most effective in inhibiting iron‐dependent lipid peroxidation in rat brain homogenates with IC50 values (12–16 µm), which is comparable with that of desferal, a prototype iron chelator that is not has orally active. Their antioxidant activities were further confirmed using electron paramagnetic resonance spectroscopy. In PC12 cell culture, the three novel chelators at 0.1 µm were able to attenuate cell death induced by serum deprivation and by 6‐hydroxydopamine. M30 possessing propargyl, the MAO inhibitory moiety of the anti‐Parkinson drug rasagiline, displayed greater neuroprotective potency than that of rasagiline. In addition, in vitro, M30 was a highly potent non‐selective MAO‐A and MAO‐B inhibitor (IC50 < 0.1 µm). However, HLA20 was more selective for MAO‐B but had poor MAO inhibition, with an IC50 value of 64.2 µm. The data suggest that M30 and HLA20 might serve as leads in developing drugs with multifunctional activities for the treatment of various neurodegenerative disorders.

Photoreduction of Carbon Dioxide to Carbon Monoxide with Hydrogen Catalyzed by a Rhenium(I) Phenanthroline−Polyoxometalate Hybrid Complex

A phenanthroline ligand decorated at the 5,6-position with a 15-crown-5 ether was used to prepare a metalorganic-polyoxometalate hybrid complex Re(I)(L)(CO)(3)CH(3)CN-MHPW(12)O(40) (L = 15-crown-5-phenanthroline, M = Na(+), H(3)O(+)). X-ray diffraction, (1)H and (13)C NMR, ESI-MS, IR, and elemental analysis were used to characterize this complex. In the presence of Pt/C, the polyoxometalate moiety in Re(I)(L)(CO)(3)CH(3)CN-MHPW(12)O(40) can oxidize H(2) to two protons and two electrons which in the presence of visible light can catalyze the photoreduction of CO(2) to CO with H(2) as the reducing agent instead of the universally used amines as sacrificial reducing agents. An EPR spectrum of a stable intermediate species under reaction conditions shows characteristics of a PW(V)W(VI)(11)O(40) and a Re(0) species with a tentative assignment of the intermediate as Re(0)(L)(CO)(3)(S)-MH(3)PW(V)W(VI)(11)O(40).

Photochemical Reduction of Carbon Dioxide Catalyzed by a Ruthenium- Substituted Polyoxometalate

A polyoxometalate of the Keggin structure substituted with Ru(III), (6)Q(5)[Ru(III)(H(2)O)SiW(11)O(39)] in which (6)Q=(C(6)H(13))(4)N(+), catalyzed the photoreduction of CO(2) to CO with tertiary amines, preferentially Et(3)N, as reducing agents. A study of the coordination of CO(2) to (6)Q(5)[Ru(III)(H(2)O)SiW(11)O(39)] showed that 1) upon addition of CO(2) the UV/Vis spectrum changed, 2) a rhombic signal was obtained in the EPR spectrum (g(x)=2.146, g(y)=2.100, and g(z)=1.935), and 3) the (13)C NMR spectrum had a broadened peak of bound CO(2) at 105.78 ppm (Delta(1/2)=122 Hz). It was concluded that CO(2) coordinates to the Ru(III) active site in both the presence and absence of Et(3)N to yield (6)Q(5)[Ru(III)(CO(2))SiW(11)O(39)]. Electrochemical measurements showed the reduction of Ru(III) to Ru(II) in (6)Q(5)[Ru(III)(CO(2))SiW(11)O(39)] at -0.31 V versus SCE, but no such reduction was observed for (6)Q(5)[Ru(III)(H(2)O)SiW(11)O(39)]. DFT-calculated geometries optimized at the M06/PC1//PBE/AUG-PC1//PBE/PC1-DF level of theory showed that CO(2) is preferably coordinated in a side-on manner to Ru(III) in the polyoxometalate through formation of a Ru-O bond, further stabilized by the interaction of the electrophilic carbon atom of CO(2) to an oxygen atom of the polyoxometalate. The end-on CO(2) bonding to Ru(III) is energetically less favorable but CO(2) is considerably bent, thus favoring nucleophilic attack at the carbon atom and thereby stabilizing the carbon sp(2) hybridization state. Formation of a O(2)C-NMe(3) zwitterion, in turn, causes bending of CO(2) and enhances the carbon sp(2) hybridization. The synergetic effect of these two interactions stabilizes both Ru-O and C-N interactions and probably determines the promotional effect of an amine on the activation of CO(2) by [Ru(III)(H(2)O)SiW(11)O(39)](5-). Electronic structure analysis showed that the polyoxometalate takes part in the activation of both CO(2) and Et(3)N. A mechanistic pathway for photoreduction of CO(2) is suggested based on the experimental and computed results.

A spectrophotometric assay for allicin, alliin, and alliinase (alliin lyase) with a chromogenic thiol: reaction of 4-mercaptopyridine with thiosulfinates

Allicin (diallylthiosulfinate) is the best known active compound of garlic. It is generated upon the interaction of the nonprotein amino acid alliin with the enzyme alliinase (alliin lyase, EC 4.4.1.4). Previously, we described a simple spectrophotometric assay for the determination of allicin and alliinase activity, based on the reaction between 2-nitro-5-thiobenzoate (NTB) and allicin. This reagent is not commercially available and must be synthesized. In this paper we describe the quantitative analysis of alliin and allicin, as well as of alliinase activity with 4-mercaptopyridine (4-MP), a commercially available chromogenic thiol. The assay is based on the reaction of 4-MP (lambda(max)=324nm) with the activated disulfide bond of thiosulfinates -S(O)-S-, forming the mixed disulfide, 4-allylmercaptothiopyridine, which has no absorbance at this region. The structure of 4-allylmercaptothiopyridine was confirmed by mass spectrometry. The method was used for the determination of alliin and allicin concentrations in their pure form as well as of alliin and total thiosulfinates concentrations in crude garlic preparations and garlic-derived products, at micromolar concentrations. The 4-MP assay is an easy, sensitive, fast, noncostly, and highly efficient throughput assay of allicin, alliin, and alliinase in garlic preparations.

Polyphenol-induced dissociation of various amyloid fibrils results in a methionine-independent formation of ROS☆

Fibrillization of amyloid polypeptides is accompanied by formation of reactive oxygen species (ROS), which, in turn, is assumed to further promote amyloid-related pathologies. Different polyphenols, all of which are established antioxidants, cause dissociation of amyloid fibrils. This study addresses the latter, poorly understood process. Specifically, we have investigated the dissociation of Abeta(42) fibrils by six different polyphenols, using electron microscopy and spectrofluorometric analysis. Simultanously, we have monitored the production of ROS using electron spin resonance (ESR) and the commercially available peroxide assay kit. Using the same methods we found that curcumin, one of the most potent destabilizing agents of Abeta(42), induced dissociation of fibrils of other amyloid polypeptides [Abeta(40), Abeta(42)Nle35, islet amyloid polypeptide and a fragment of alpha-synuclein]. When the solution contained traces of transition metal, all the dissociation reactions were accompanied by ROS formation, independent of the presence of a methionine residue. Kinetic studies show that the formation of ROS lags behind dissociation, indicating that if casual relationship exists between these two processes, then ROS formation may be considered a consequence and not a cause of dissociation. These findings open new avenues in amyloid research that will be required to gain further understanding of our results and of their implications.

EPR studies of hypericin. Photogeneration of free radicals and superoxide

Hypericin, a potent antiviral agent, displays, in the absence of light and electron donors, an EPR signal which is attributed to a semiquinone-like radical, formed by intermolecular electron transfer. On irradiation with visible light the amplitude of the EPR signal increases significantly. This increase is highest (ca. 20 fold) in aqueous dispersions of the lysine salt of hypericin. Irradiation of hypericin water aggregates in the presence of oxygen generates superoxide radicals which may be registered by the spin trap technique.This finding implies that the free radicals of hypericin and superoxide radicals formed on photoirradiation of hypericin may play a hitherto unrecognized role in the biological activities elicited by hypericin both in vivo and in vitro.