Absence Of Peroxide Research Articles

Interactions between Drosophila and its natural yeast symbionts-Is Saccharomyces cerevisiae a good model for studying the fly-yeast relationship?

Yeasts play an important role in the biology of the fruit fly, Drosophila melanogaster. In addition to being a valuable source of nutrition, yeasts affect D. melanogaster behavior and interact with the host immune system. Most experiments investigating the role of yeasts in D. melanogaster biology use the baker’s yeast, Saccharomyces cerevisiae. However, S. cerevisiae is rarely found with natural populations of D. melanogaster or other Drosophila species. Moreover, the strain of S. cerevisiae used most often in D. melanogaster experiments is a commercially and industrially important strain that, to the best of our knowledge, was not isolated from flies. Since disrupting natural host–microbe interactions can have profound effects on host biology, the results from D. melanogaster–S. cerevisiae laboratory experiments may not be fully representative of host–microbe interactions in nature. In this study, we explore the D. melanogaster-yeast relationship using five different strains of yeast that were isolated from wild Drosophila populations. Ingested live yeasts have variable persistence in the D. melanogaster gastrointestinal tract. For example, Hanseniaspora occidentalis persists relative to S. cerevisiae, while Brettanomyces naardenensis is removed. Despite these differences in persistence relative to S. cerevisiae, we find that all yeasts decrease in total abundance over time. Reactive oxygen species (ROS) are an important component of the D. melanogaster anti-microbial response and can inhibit S. cerevisiae growth in the intestine. To determine if sensitivity to ROS explains the differences in yeast persistence, we measured yeast growth in the presence and absence of hydrogen peroxide. We find that B. naardenesis is completely inhibited by hydrogen peroxide, while H. occidentalis is not, which is consistent with yeast sensitivity to ROS affecting persistence within the D. melanogaster gastrointestinal tract. We also compared the feeding preference of D. melanogaster when given the choice between a naturally associated yeast and S. cerevisiae. We do not find a correlation between preferred yeasts and those that persist in the intestine. Notably, in no instances is S. cerevisiae preferred over the naturally associated strains. Overall, our results show that D. melanogaster-yeast interactions are more complex than might be revealed in experiments that use only S. cerevisiae. We propose that future research utilize other yeasts, and especially those that are naturally associated with Drosophila, to more fully understand the role of yeasts in Drosophila biology. Since the genetic basis of host–microbe interactions is shared across taxa and since many of these genes are initially discovered in D. melanogaster, a more realistic fly-yeast model system will benefit our understanding of host–microbe interactions throughout the animal kingdom.

Role of Active Site Residues on Catalytic Activity of Catalase with Oxidase Activity from Scytalidium Thermophilum

Scytalidium thermophilum produces a catalase with phenol oxidase activity (CATPO) that catalyses the dismutation of hydrogen peroxide (H2O2) to dioxygen and water and also oxidizes several phenolic compounds in the absence of hydrogen peroxide. It comprises 717 amino acids with a 19 amino acid signal sequence, and a 17 amino acid prosequence. It is a homotetrameric protein of molecular mass 320 kDa and subunit molecular mass 80 kDa. Although catalases have been studied for many years, a peroxide independent oxidative activity of catalases has recently been recognized. There are a great number of reports available describing the structural and biochemical characterization of catalases. However basic questions related to substrate and product flow remain unanswered, particularly related to the oxidase activity. The goals of our current studies are to investigate the main and lateral channels known that connect the deeply buried active site to the exterior of the enzyme. We have introduced a number of mutations into these regions and analyzed their specific activities.

Multifunctional gold nanoparticles as signal transducers for fabrication of 1:2 molecular demultiplexer.

A label-free and enzyme-free demultiplexer system for the fabrication of 1:2 molecular demultiplexer with luminol functionalized gold nanoparticles (Lum-AuNPs) as signal transducers was developed for the first time. The Lum-AuNPs had both chemiluminescence (CL) activity and surface plasmon resonance property. It was found that organothiols (RSH) could easily induce the aggregation of AuNPs via strong Au-S covalent interactions in the absence of hydrogen peroxide (H2O2), generating a red shift in the absorption band of AuNPs. However, the presence of H2O2 would readily oxidize RSH to disulfide (RS-SR), and the aggregation of Lum-AuNPs did not occur due to lack of the sulfhydryl group. Meanwhile, H2O2 could react with Lum-AuNPs, producing a strong CL emission owing to the enhancement effect of RSH on AuNPs-luminol-H2O2 CL system. Thus, RSH, H2O2, absorbance ratio, and CL intensity served as the signal input, address input, and two different signal outputs of the 1:2 molecular demultiplexer, respectively.

Seven-coordinated cobalt(II) complexes with 2,6-diacetylpyridine bis(4-hydroxybenzoylhydrazone): synthesis, characterisation, DNA binding and cleavage properties

Synthesis and characterisation of three seven-coordinated cobalt(II) complexes of 2,6-diacetylpyridine bis(4-hydroxybenzoylhydrazone) (H4L) ligand, [Co(H2L)(H2O)2] (1), [Co(H4L)(N3)2] (2) and [Co(H4L)(NCS)2] (3) are described. The structures of the complexes were characterised by elemental analysis, IR, UV–vis and magnetic susceptibility measurement. The molecular structure of the [Co(H4L)(NCS)2] (3) was also determined by X-ray crystallography. Single crystal X-ray revealed that the Co(II) complex (3) has a pentagonal–bipyramidal coordination geometry, with pentadentate N3O2 ligand in the equatorial plane of the bipyramid and two isothiocyanato groups in the axial area. Interaction of the cobalt(II) complexes with CT-DNA was investigated by absorption titration method and viscosity measurements. Cleavage activity of the complexes with pBR 322 plasmid DNA was evaluated by agarose gel electrophoresis in presence and absence of an oxidative agent, and the mechanism of DNA cleavage was investigated. The results suggest that the cobalt(II) complexes bind effectively and they exhibit nuclease activity, which has strong dependence on the concentration of complex and reaction time, both in presence and absence of hydrogen peroxide.

Copper(II) complexes with new fluoroquinolones: Synthesis, structure, spectroscopic and theoretical study, DNA damage, cytotoxicity and antiviral activity

Copper(II) complexes with fluoroquinolones in the presence of the nitrogen donor heterocyclic ligands 1,10-phenanthroline have been considered in detail. The phenanthroline moiety was introduced into the ligand environment with the aim to determine whether the nuclease activity is feasible. All suitable X-ray structures of the complexes under study reveal a distorted square pyramidal coordination geometry for Cu(II) atom. The conformational and spectroscopic (FT-IR and UV–visible) behavior has been analyzed and has been interpreted with respect to B3LYP/6-311G* calculations including molecular dynamics. The ability of the complexes to cleave DNA was studied by agarose gel electrophoresis with plasmid DNA pBSK+. The results have confirmed that the complexes under study behave as the chemical nucleases. Nuclease like activity in the absence of hydrogen peroxide allows us to deduce an interaction of the complexes with the DNA resulting in the conversion of supercoiled circular DNA to the nicked form. The DNA cleavage activity enhanced by the presence of hydrogen peroxide demonstrates the participation of reactive oxygen species, such as superoxide radical anions and hydroxyl radicals which presence was confirmed independently using the standard radical scavenging agents. It has been suggested that the radical formation through the Fenton/Haber–Weiss reaction is mediated by the redox cycling mechanisms with the participation of cupric/cuprous ions. Cytotoxic activity was evaluated as the 50% cytotoxic concentration (CC50). The potential effects of tested compounds on replication of murine gammaherpesvirus 68 (MHV-68) under in vitro conditions were also evaluated. However, no antiviral activity against MHV-68 was observed.

Photodegradation of single and mixture of parabens – Kinetic, by-products identification and cost-efficiency analysis

Esters of p-hydroxybenzoic acid, commonly known as parabens are preservatives used in a variety of personal care products, cosmetics, pharmaceutical and food products.This paper presents the results of studies on photochemical degradation of hazardous water contaminants – methyl- (MP), ethyl- (EP), propyl- (PP), butyl- (BuP), benzylparaben (BeP) and p-hydroxybenzoic acid (p-HBA) (individually or in mixture) using ultraviolet C lamps in the presence and absence of hydrogen peroxide. Both systems were very efficient on the degradation of the single parent compounds. Total removal of individual pollutant was achieved after 2h or 6min for UVC and UVC/H2O2, respectively. The kinetic constants of hydroxyl radicals reaction and the photolysis quantum yields were obtained for each pollutant. The decomposition of the mixture of the six compounds with the H2O2/UVC system ensures a complete degradation after 30min with a degree of mineralization of 25%. Whereas only 10% of mineralization was observed after the same time for photolysis. The pseudo-first-order fluence-based rate constants (for UVC and H2O2/UVC) for each paraben in the mixture reaction were calculated for the first time. The analysis using UHPLC/MS/MS technique shows that hydroxylation is the main pathway of the reaction with hydroxyl radicals. In fact, p-hydroxybenzoic acid, 2,4-dihydroxybenzoic acid and 3,4-dihydroxybenzoic acid were identified as a major transformation products. Moreover the mono, di and tri derivatives of parabens were detected as the main intermediates formed during oxidation. The fluence-based experimental data and cost analysis showed that the H2O2/UVC treatment was effective in mineralizing of the parabens mixture.

Seven coordinated cobalt(II) complexes with 2,6-diacetylpyridine bis(4-acylhydrazone) ligands: Synthesis, characterization, DNA-binding and nuclease activity

Abstract A new series of pentadentate 2,6-diacetylpyridine bis(4-acylhydrazone)s (H2L1 and H2L2) based seven-coordinated cobalt(II) complexes, [Co(Ln)X2] (n = 1 and X = DMF for (1); n = 2 and X = H2O for (2)); [Co(H2Ln)Y2] (n = 1 or 2; Y = N3− or NCS−), has been synthesized and characterized by using spectroscopic techniques. Single crystal X-ray study of [Co(L1)(DMF)2] (1) complex exhibits pentagonal–bipyramidal coordination geometry where the pentadentate N3O2 ligand in the equatorial plane of the bipyramid and two dimethylformamide molecules in the axial area. Interaction of the cobalt(II) complexes with CT DNA has been investigated by absorption titration method and viscosity measurements which reveal that the cobalt(II) complexes could bind with CT DNA through intercalation. Cleavage activity of the complexes (1) and (2) with pBR 322 plasmid DNA was evaluated by agarose gel electrophoresis demonstrating that the ability of the complexes to cleave the pBR 322 plasmid DNA via oxidative pathway, possibly due to the involvement of a diffusible hydroxyl radical mechanism in presence and absence of an oxidative agent. The nuclease activity of the Co(II) complexes has strong dependence on the concentration of complex and reaction time, both in presence and absence of hydrogen peroxide.

Photocatalysis of TiO2 – Supported Graphene Oxide and its Reduced Form towards Phenol Degradation

Commercial nanocrystals graphite was oxidized to graphene oxide (GO) using Hummers’ method. Then, GO was directly impregnated mechanically over TiO2 (GOTi) and reduced later using a stream of hydrogen gas in a controlled reactor at 450 oC (H2RGOTi) or reduced initially by hydrazine hydrate solution (HH) in a 1000-W microwave oven then loaded later on TiO2 (HHRGOTi). Also, hydrothermal reduction was applied to obtain reduced GO/TiO2 (RGOTi). X-ray photoelectron spectroscopy (XPS), Raman spectra and X-ray diffraction patterns (XRD) confirmed the oxidation and reduction processes of graphite and GO respectively. XPS have revealed that GO surface consists mainly of hydroxyl, epoxy, carbonyl and carboxylic groups. From argon bombardment etching, it was revealed that GO is on the surface of TiO2. Catalysts were prepared and tested for photodegradation of phenol under UV illumination in presence and absence of hydrogen peroxide. In addition, the photocatalytic activity of the as-prepared catalysts was compared with commercial TiO2. It was concluded that the 0.1-RGOTi catalyst has the best performance in terms of phenol degradation under UV illumination in absences and presence of H2O2. However, its performance is better in presence of H2O2 especially at small time intervals.

Carotene-degrading activities from Bjerkandera adusta possess an application in detergent industries.

Four extracellular enzymes, a versatile peroxidase, a manganese peroxidase, a dye-decolorizing peroxidase and a lignin peroxidase were discovered in liquid cultures of the basidiomycete Bjerkandera adusta. All of them cleaved β-carotene effectively. Expression was enhanced in the presence of β-carotene or Coomassie Brilliant Blue and peaked after 7-9 days. The monomeric proteins were purified by ion exchange and size exclusion chromatography and exhibited molecular masses of 41, 43, 51 and 43 kDa, respectively. The coding sequences showed homologies from 61 to 89 % to peroxidases from other basidiomycetes. The novel enzymes retained strong activity even in the absence of hydrogen peroxide and at alkaline pH. De-staining of fabrics using detergent-tolerant enzymes may help to save the most important bio-resources, energy and water, in washing processes and led to green processes in textile cleaning.

Modulatory effects of green tea on HEK-­293 cell energy metabolism: implications in diabetic nephropathy

The consumption of green tea is epidemiologically associated with a reduction in diabetic nephropathy; however its prophylactic effect remains unclear in an oxidative stress-­‐associated diabetic milieu. The energy metabolism of HEK-­‐293 cell, pretreated with variable concentrations of green tea, was evaluated under different hydrogen peroxide (H 2 O 2 ) concentrations using the MTT assay. Green tea modulated the energy metabolism in renal cell line under different hydrogen peroxide challenge. In the absence of hydrogen peroxide, green tea at concentrations of 0.25 mg/mL and 0.50 mg/mL significantly increased the energy metabolism of HEK-­‐293 cell by 81.5 % (P < 0.01) and 56.1 % (P < 0.05), respectively. Green tea at a concentration of 3 mg/mL significantly reduced (P < 0.05) the energy metabolism in HEK-­‐293 cell by 36.6 % and 20.7 % when incubated in the presence of 200 μM and 500 μM H 2 O 2 , respectively. At high concentration, H 2 O 2 and green tea have shown the ability in reducing the energy metabolism of HEK-­‐293 cell. Conclusively, moderate consumption of green tea could form part of a healthy lifestyle that might ameliorate features of metabolic syndrome and subsequent risks for diabetic nephropathy, an outcome that can be determined by further clinical studies. KEY WORDS : Diabetes; Green tea and wellness; Antioxidant; Nephropathy; Metabolic syndrome

A study on prophagic and chromosomal sodC genes involvement in Escherichia coli O157:H7 biofilm formation and biofilm resistance to H2O2.

Escherichia coli O157:H7 possesses one chromosomal and two prophagic sodC genes encoding for Cu,Zn superoxide dismutases. We evaluated the contribution of sodC genes in biofilm formation and its resistance to hydrogen peroxide. The biofilm of sodC deletion mutants has been studied, in presence or absence of hydrogen peroxide, by crystal violet in 96-well plates and Scanning Electron Microscopy on glass coverslips. Deletion of prophagic sodC genes had no effect on biofilm construction, in contrast to the chromosomal gene deletion. Hydrogen peroxide treatment showed higher cell mortality and morphological alterations in sodC deletion mutants respect to wild type. These effects were related to the biofilm development stage. The role of the three SodCs is not redundant in biofilm formation and the resistance to oxidative damage. The stage of biofilm development is a crucial factor for an effective sanitization.

Influence of electrode material on hydrogen peroxide generation by DC pinhole discharge

Abstract In this work, several materials were studied as electrodes in a pinhole configuration of a DC plasma discharge to estimate their effect on the efficiency of the discharge, indicated by hydrogen peroxide production. Detection was carried out using a specific titanium reagent. This was combined with ICP-OES analysis of the final solutions to determine the difference between the amount of electrode material released during the discharge operation and electrolysis experiment carried out under the same conditions. It was found that from seven studied electrode materials, graphite gives the best results, while lower cost aluminum and titanium-zinc still work well. The most unsuitable materials were copper and brass; in these cases, no hydrogen peroxide was detected in the cathode part of the reactor. Results obtained by ICP analysis indicate that even in the case of brass, the absence of hydrogen peroxide is due to the presence of copper in the material. It probably affects both directly the phase of discharge creation and propagation and the decomposition reactions.

Performance Improvement of Hydrogen Peroxide Sensor via Cadmium Doped Nickel Oxide Modifying N-Silicon Electrode

Cadmium ion doping was carried out during electrochemically cathodic plating nickel hydroxide on platinum films coated n-silicon (Pt/n-n+-Si electrode) in 0.1 M nickel nitrate solution containing 0.035 M cadmium nitrate. The morphology and composition of the products were characterized by scanning electron microscope (SEM) and x-ray photoelectron spectroscopy (XPS), respectively. A two-electrode cell based on Cd-doped NiO/Pt/n-n+-Si electrode and a platinum counter has been used for determination of hydrogen peroxide in absence of reference electrode by photocurrent measurement at a zero bias. The emphasis is laid on that the cadmium doping remarkably improves the sensibility of the photoelectrochemical hydrogen peroxide sensor. Under optimizing conditions a sensitivity of 254.6 μA mM-1 cm-2 and a linear response range from 0.02 mM up to 0.12 mM with a determination limit of 2.0 μM were achieved in a potassium hydroxide (KOH) solution at pH =13.3.

Oxidative stress-induced JNK1 phosphorylation inhibits hedgehog signalling and osteoblast differentiation

Oxidative stress has been progressively recognised as a key player in the development of osteoporosis. Although oxidative stress induces osteoporosis partly through inhibition of hedgehog signalling, the details of the signal interaction remain unclear. To elucidate this mechanism, we investigated osteoblast differentiation in C3H10T1/2 cells, murine embryonic mesenchymal cells, with recombinant sonic hedgehog (shh) in the presence or absence of hydrogen peroxide, and examined Gli transcriptional activity, with a focus on the interaction with mitogen-activated protein kinases (MAPKs) as possible mediators. The transcription of Gli1, Ptch1b, Alp and Bsp was facilitated by shh and strongly inhibited by oxidative stress with the hydrogen peroxide treatment; however, the other Gli isoforms, Bmp2, Runx2 and Osx, were unaffected. Oxidative stress also facilitated the phosphorylation of JNK1 and p38. Anisomycin treatment as a JNK activator or transient overexpression of the constitutive JNK1 active form inhibited hedgehog activity. Interestingly, the inhibitory effect of hydrogen peroxide on hedgehog signalling was blocked by a JNK inhibitor, but not by a p38 inhibitor. Osteoblast differentiation determined by calcium staining was, in part, rescued from oxidative stress by the JNK inhibitor. These findings suggest that oxidative stress inhibits hedgehog signalling and subsequent osteoblast differentiation through an interaction between phospho-JNK1 and GLI1.

Synthesis and crystal structures of sterically tuned ether functionalized NHC–silver(I) complexes: antibacterial and nucleic acid interaction studies

A series of new imidazolium salts (1–4) as N-heterocyclic carbene (NHC) precursors have been synthesized by successive N-alkylation method. Reactions of these salts with Ag2O by varying the metal to salt ratio forms a series of new Ag(I)–NHC complexes (5–8). All compounds were characterized by physico-chemical and spectroscopic techniques. The molecular structures of 1 and 5 were characterized by single-crystal X-ray diffraction analysis. A comparative investigation of the bacterial growth inhibition potential of the salts and respective complexes indicates that 5–8 displayed good antibacterial activities on Staphylococcus aureus (ATCC 12600) and Escherichia coli (ATCC 11303) compared with the salts. Furthermore, it was observed that with increase in chain length at N-positions, the antibacterial activities also increased. Nuclease activity of the reported salts and Ag(I)–NHC complexes with nucleic acids (DNA and RNA) were also studied using agarose gel electrophoresis; the results show that the compounds do not have any apparent interaction with nucleic acids in the absence of hydrogen peroxide (H2O2). However, 5 and 8 were efficient in promoting the cleavage of nucleic acids in the presence of H2O2.

UV VIS Studies of Ce(III)/Ce(IV) Redox Reactions to Understand Abrasive and Work Surface Interactions during STI CMP

In the current fabrication processes for integrated chips (IC), shallow trench isolation (STI) scheme is used to isolate and insulate transistors. Chemical mechanical planarization (CMP) is a key step in STI integration scheme. High selectivity slurries (HSS) which yield high silicon dioxide to silicon nitride polish rate are often used, and this is achieved by controlling the solid loading and the chemical concentration of additives [1]. Ceria based slurries with suitable additives are known to provide high selectivity [2, 3, 5]. Ceria is known to interact chemically with the silicon dioxide surface during CMP [2,3] and it is proposed that Ce(III) might play a role in the chemical interaction [4], but the exact nature of the species interacting with oxide are not known.Addition of hydrogen peroxide (H2O2) to ceria based slurries is reported to suppress the oxide and nitride removal rates [4]. However, when other abrasives are employed, hydrogen peroxide does not modify the removal rate significantly [4]. Ce (III) and Ce (IV) ions are known to exhibit UV VIS absorption peak at different wavelength regions.In this work a study on the kinetics of Ce (III) and Ce (IV) ions in the presence of H2O2is undertaken using UV-Visible Spectroscopy. Ce (III) chloride and Ce (IV) sulfate are employed for the initial study and the effect of hydrogen peroxide on changing the equilibrium composition is analyzed. The absorption is also monitored as a function of pH and time, to understand the dynamics of the system. Further, ceria abrasives are employed in the study and the nature of the species dissolving into the aqueous medium in presence of and absence of hydrogen peroxide, at different pH values are analyzed. The results show that the equilibrium between Ce (III) and Ce (IV) is shifted by the hydrogen peroxide which may help explain the suppression of oxide and nitride CMP rate by the addition of peroxide to ceria based slurries.

Degradation of ethylenethiourea pesticide metabolite from water by photocatalytic processes

In this study, photocatalytic (photo-Fenton and H2O2/UV) and dark Fenton processes were used to remove ethylenethiourea (ETU) from water. The experiments were conducted in a photo-reactor with an 80 W mercury vapor lamp. The mineralization of ETU was determined by total organic carbon analysis, and ETU degradation was qualitatively monitored by the reduction of UV absorbance at 232 nm. A higher mineralization efficiency was obtained by using the photo-peroxidation process (UV/H2O2). Approximately 77% of ETU was mineralized within 120 min of the reaction using [H2O2]0 = 400 mg L−1. The photo-Fenton process mineralized 70% of the ETU with [H2O2]0 = 800 mg L−1 and [Fe2+] = 400 mg L−1, and there is evidence that hydrogen peroxide was the limiting reagent in the reaction because it was rapidly consumed. Moreover, increasing the concentration of H2O2 from 800 mg L−1 to 1200 mg L−1 did not enhance the degradation of ETU. Kinetics studies revealed that the pseudo-second-order model best fit the experimental conditions. The k values for the UV/H2O2 and photo-Fenton processes were determined to be 6.2 × 10−4 mg L−1 min−1 and 7.7 × 10−4 mg L−1 min−1, respectively. The mineralization of ETU in the absence of hydrogen peroxide has led to the conclusion that ETU transformation products are susceptible to photolysis by UV light. These are promising results for further research. The processes that were investigated can be used to remove pesticide metabolites from drinking water sources and wastewater in developing countries.

DNA damage by oxo- and peroxo-chromium(v) complexes: insight into the mutation and carcinogenesis mechanisms

The carcinogenic and mutagenic properties of chromium(vi) compounds are attributed to hypervalent chromium species, especially metastable oxo- and peroxo-chromium(v)/(iv) complexes. To understand the nature of DNA damage induced by various hypervalent chromium complexes, reactions of sodium bis(2-ethyl-2-hydroxybutanato)oxochromate(v) (I) with single- and double-stranded DNA were investigated in the presence of hydrogen peroxide and a radical scavenger. In these reactions, three DNA damaging agents, oxo- and peroxo-chromium(v) complexes and hydroxyl radicals, were generated. Both double and single-stranded DNA cleavages were largely observed after piperidine treatment, indicating base-labile scissions. Although these cleavages appeared to be random in most cases, a modest selectivity towards the G base was observed for the double stranded oligonucleotides. A variety of sugar and base oxidation products were detected after enzymatic digestion of the reaction mixtures with exo- and endo-nucleases. These products include furfural, 5-methylene furanone, 8-oxo-7,8-dihydro-2′-guanosine (8-oxodGuo), thymine propenal, deoxyribonucleoside derivative of 5-guanidinohydantoin (dGh), cytosine glycol and 5-hydroxy cytosine. The first three mentioned products were observed for reactions of compound I with DNA in the absence of peroxide. Since compound I generated a persistent peroxo-chromium(v) complex in situ and oxidation of DNA by the oxo-chromium(v) was relatively slow, it appears that the peroxo-species was largely responsible for the formation of dGh through a four-electron oxidation of the guanine residue following the mechanism proposed by Meunier and coworkers. Likewise, the formation of propenals follows a similar pathway observed for bleomycine-mediated DNA oxidation that follows Hecht's mechanism. On the other hand, 5,6-dihydroxy-5,6-dihydro-2′-deoxycytidine (dCydGl) and 5-hydroxy-2′-deoxycytidine (5-OHdCyd) were most likely formed from the reactions of DNA with the hydroxyl radical. We hypothesize that base oxidations and the formation of abasic sites are most likely responsible for transverse DNA mutation. Reactions of DNAs with a chromium(v)-peroxo complex, generated in situ, yielded both sugar and base oxidation products including guanidinohydantoin.

One-electron oxidation of diclofenac by human cytochrome P450s as a potential bioactivation mechanism for formation of 2′-(glutathion-S-yl)-deschloro-diclofenac

Reactive metabolites have been suggested to play a role in the idiosyncratic hepatotoxicity observed with diclofenac (DF). By structural identification of the GSH conjugates formed after P450-catalyzed bioactivation of DF, it was shown that three types of reactive intermediates were formed: p-benzoquinone imines, o-imine methide and arene-oxide. Recently, detection of 2′-(glutathion-S-yl)-deschloro-diclofenac (DDF-SG), resulting from chlorine substitution, suggested the existence of a fourth type of P450-dependent reactive intermediate whose inactivation by GSH is completely dependent on presence of glutathione S-transferase. In this study, fourteen recombinant cytochrome P450s and three flavin-containing monooxygenases were tested for their ability to produce oxidative DF metabolites and their corresponding GSH conjugates. Concerning the hydroxymetabolites and their GSH conjugates, results were consistent with previous studies. Unexpectedly, all tested recombinant P450s were able to form DDF-SG to almost similar extent. DDF-SG formation was found to be partially independent of NADPH and even occurred by heat-inactivated P450. However, product formation was fully dependent on both GSH and glutathione-S-transferase P1-1. DDF-SG formation was also observed in reactions with horseradish peroxidase in absence of hydrogen peroxide. Because DDF-SG was not formed by free iron, it appears that DF can be bioactivated by iron in hemeproteins. This was confirmed by DDF-SG formation by other hemeproteins such as hemoglobin. As a mechanism, we propose that DF is subject to heme-dependent one-electron oxidation. The resulting nitrogen radical cation, which might activate the chlorines of DF, then undergoes a GST-catalyzed nucleophilic aromatic substitution reaction in which the chlorine atom of the DF moiety is replaced by GSH.

Imaging catalase reactions through interactions between liquid crystals and oil-in-water emulsions

In this study, a simple label-free technique for imaging catalase reactions through interactions between liquid crystals (LCs) and oil-in-water emulsions was developed. We prepared a dodecanal-in-water emulsion, and mixed the emulsion with 4-cyano-4′-pentylbi-phenyl (5CB) droplets. In the absence of hydrogen peroxide, a dark image was observed between crossed polarisers because the 5CB and dodecanal droplets coalesced resulting in the formation of a population of droplets comprised of 5CB and dodecanal that are isotropic. In contrast, if hydrogen peroxide was present, a bright appearance was observed, because some fraction of the dodecanal was converted to dodecanoic acid, which prevented coalescence of 5CB and dodecanal, and thus the 5CB droplets retained their nematic order. Since catalase could decompose hydrogen peroxide, we observed a dark image after LC made contact with a dodecanal–hydrogen peroxide mixture that had been pre-incubated with catalase. Control experiments were conducted to confirm the specificity of the enzymatic reaction. The detection limit of catalase in our system was 0.1 μg/mL. These results demonstrate that LC with oil-in-water emulsion can be used for the detection of relevant enzymatic reactions.