Characterization of oxidative stress in Leishmaniasis-infected or LPS-stimulated macrophages using electrochemical impedance spectroscopy

The physiological changes caused by external stimuli can be employed as parameters to study pathogen infection in cells and the effect of drugs. Among analytical methods, impedance is potentially useful to give insight into cellular behavior by studying morphological changes, alterations in the physiological state, production of charged or redox species without interfering with in vitro cellular metabolism and labeling. The present work describes the use of electrochemical impedances spectroscopy to simply monitor by modeling impedance plots (Nyquist diagram) in appropriate equivalent circuit, the changes affecting murine macrophage cell line (RAW 264.7) in response to parasite infection by Leishmania amazonensis or to lipopolysaccharide (LPS) treatment. These results demonstrate the ability of electrochemical impedance spectroscopy to discriminate between two opposite cell responses associated to two different stimuli, one caused by the internalization of a parasite, and the other by activation by a bacterium component. Indeed, the study has allowed the characterization, from an electrical point of view, of the extra-cellular NO radical produced endogenously and in great quantities by the inducible form of NO-synthase in the case of LPS-stimulatedmacrophages. This production was not observed in the case of Leishmania-infectedmacrophages for which to survive and multiply, the parasite itself possesses mechanisms which may interfere with NO production. In this latest case, only the intracellular production of ROS was observed. To confirm these interpretations confocal microscopy analysis using the ROS (reactive oxygen species) fluorescent probe 2′,7′-dichlorodihydrofluorescein diacetate and electron paramagnetic resonance experiments using Fe(DETC)2 as NO radical spin trap were carried out

Electrochemical impedance spectroscopy to study physiological changes affecting the red blood cell after invasion by malaria parasites

The malaria parasite, Plasmodium falciparum, invades human erythrocytes and induces dramatic changes in the host cell. The idea of this work was to use RBC modified electrode to perform electrochemical impedance spectroscopy (EIS) with the aim of monitoring physiological changes affecting the erythrocyte after invasion by the malaria parasite. Impedance cell-based devices are potentially useful to give insight into cellular behavior and to detect morphological changes. The modelling of impedance plots (Nyquist diagram) in equivalent circuit taking into account the presence of the cellular layer, allowed us pointing out specific events associated with the development of the parasite such as (i) strong changes in the host cell cytoplasm illustrated by changes in the film capacity, (ii) perturbation of the ionic composition of the host cell illustrated by changes in the film resistance, (iii) releasing of reducer (lactic acid or heme) and an enhanced oxygen consumption characterized by changes in the charge transfer resistance and in the Warburg coefficient characteristic of the redox species diffusion. These results show that the RBC-based device may help to analyze strategic events in the malaria parasite development constituting a new tool in antimalarial research

Electrochemical behavior of indolone-N-oxides: Relationship to structure and antiplasmodial activity

Indolone-N-oxides exert high parasiticidal activity at the nanomolar level in vitro against Plasmodiumfalciparum, the parasite responsible for malaria. The bioreductive character of these molecules was investigated using cyclic voltammetry and EPR spectroelectrochemistry to examine the relationship between electrochemical behavior and antimalarial activity and to understand theirmechanisms of action. For all the compounds (37 compounds) studied, the voltammograms recorded in acetonitrile showed a well-defined and reversible redox couple followed by a second complicated electron transfer. The first reduction (−0.88 VbE1/2b−0.50 V vs. SCE) was attributed to the reduction of the N-oxide function to form a radical nitroxide anion. The second reduction (−1.65 VbE1/2b−1.14 V vs. SCE) was assigned to the reduction of the ketone function. By coupling electrochemistry with EPR spectroscopy, the EPR spectra confirmed the formation of the nitroxide anion radical.Moreover, the experiments demonstrated that a slowprotonation occurs at the carbon of the nitrone function and not at the NO function. A relationship between electrochemical behavior and indolone-N-oxide structure can be established for compounds with R1=―OCH3, R2=H, and electron-withdrawing substituents on the phenyl group at R3. The results help in the design of new molecules with more potent in vivo antimalarial activity

Pro-oxidant properties of indolone-N-oxides in relation to their antimalarial properties

Indolone-N-oxides (INODs) are bioreducible and possess remarkable anti-malarial activities in the low nanomolar range in vitro against different Plasmodium falciparum (P. falciparum) strains and in vivo. INODs have an original mechanism of action: they damage the host cell membrane without affecting non-parasitized erythrocytes. These molecules produce a redox signal which activates SYK tyrosine kinases and induces a hyperphosphorylation of AE1 (band 3, erythrocyte membrane protein). The present work aimed to understand the early stages of the biochemical interactions of these compounds with some erythrocyte components from which the redox signal could originate. The interactions were studied in a biomimetic model and compared with those of chloroquine and artemisinin. The results showed that INODs i) do not enter the coordination sphere of the metal in the heme iron complex as does chloroquine; ii) do not generate iron-dependent radicals as does artemisinin; iii) generate stable free radical adducts after reduction at one electron; iv) cannot trap free radicals after reduction. These results confirm that the bioactivity of INODs does not lie in their spin-trapping properties but rather in their pro-oxidant character. This property may be the initiator of the redox signal which activates SYK tyrosine kinases

Concentration and purification by magnetic separation of the erythrocytic stages of all human Plasmodium species

International audienceBackground : Parasite concentration methods facilitate molecular, biochemical and immunologicalresearch on the erythrocytic stages of Plasmodium. In this paper, an adaptation of magnetic MACS®columns for the purification of human Plasmodium species is presented. This method was useful forthe concentration/purification of either schizonts or gametocytes.Results and conclusions : The magnetic removal of non-parasitized red blood cells (in vivo andin vitro) using magnetic columns (MACS) was evaluated. This easy-to-use technique enrichedschizonts and gametocytes from Plasmodium falciparum in vitro cultures with a very high degree ofpurity. In addition, all haemozoin-containing stages (schizonts and/or gametocytes) from theperipheral blood of infected patients could be concentrated using this method. This method isparticularly useful for the concentration of non-falciparum species, which do not grow in cultureand are otherwise difficult to obtain in large amounts

Antioxidant Capacity of Cotyledons and Germs of Soybeanb in Relation to Their Isoflavone Content

Svrha je ovog istraživanja bila proučiti odnos antioksidativnog kapaciteta i udjela izoflavona u ekstraktu soje s obzirom na geografsko podrijetlo i kultivar. Uzorci soje uzeti su iz dva dijela zrna soje, klice i kotiledona, s dvaju zemljopisnih lokacija (L1 i L2) i iz dva kultivara (Queen i Imari), ukupno 8 različitih uzoraka. HPLC metoda potvrdila je veći udio izoflavona u klicama nego u kotiledonima, i to u uzorcima s lokacije L2 i u kultivaru Queen. Antioksidativni kapacitet uzoraka soje određen je dvjema metodama, uklanjanjem 2,2\u27- difenil-1-pikrilhidrazil radikala i određivanjem sposobnosti apsorpcije kisikovih radikala. Rezultati obiju metoda pokazali su veću antioksidativnu aktivnost ekstrakta klice od ekstrakta kotiledona.The aim was to study the relationship between the antioxidant capacity and the isoflavone content of soybean extracts depending on both geographic origin and cultivar. Soybean samples were obtained from two soybean seed parts, germ and cotyledon, from two geographical locations (L1, L2) and two cultivars (Queen, Imari), which gave 8 different samples. HPLC determination confirmed higher isoflavone content in germs than in cotyledons, with higher contents in site L2, and in the Queen cultivar. The antioxidant capacity of soybean samples was determined with two methods, the 2,2-diphenyl-1-picrylhydrazyl scavenging assay and the oxygen radical absorbance capacity assay. The results obtained with both assays showed differences in antioxidant capacity between germ and cotyledon extracts, with a higher antioxidant activity of germ extracts

Radical trapping properties of imidazolyl nitrones

The ability of ten imidazolyl nitrones to directly scavenge free radicals (R√) generated in polar (√OH, cysteinyl, √CH3) or in apolar (CH3–√CH–CH3) media has been studied. When oxygen or sulfur-centered radicals are generated in polar media, EPR spectra are not or weakly observed with simple spectral features. Strong line intensities and more complicated spectra are observed with the isopropyl radical generated in an apolar medium. Intermediate results are obtained with √CH3 generated in a polar medium. EPR demonstrates the ability of these nitrones to trap radicals to the nitrone C(α) atom (alpha radical adduct) and to the imidazol C(5) atom (5-radical adduct). Beside the nucleophilic addition of the radical to the C(α) atom, the EPR studies suggest a two-step mechanism for the overall reaction of R√ attacking the imidazol core. The two steps seem to occur very fast with the √OH radical obtained in a polar medium and slower with the isopropyl radical prepared in benzene. In conclusion, imidazolyl nitrones present a high capacity to trap and stabilize carbon-centered radicals

NQO2 is a reactive oxygen species generating off-target for acetaminophen

[Image: see text] The analgesic and antipyretic compound acetaminophen (paracetamol) is one of the most used drugs worldwide. Acetaminophen overdose is also the most common cause for acute liver toxicity. Here we show that acetaminophen and many structurally related compounds bind quinone reductase 2 (NQO2) in vitro and in live cells, establishing NQO2 as a novel off-target. NQO2 modulates the levels of acetaminophen derived reactive oxygen species, more specifically superoxide anions, in cultured cells. In humans, NQO2 is highly expressed in liver and kidney, the main sites of acetaminophen toxicity. We suggest that NQO2 mediated superoxide production may function as a novel mechanism augmenting acetaminophen toxicity

Dereplication of natural products from complex extracts by regression analysis and molecular networking: case study of redox-active compounds from Viola alba subsp. dehnhardtii

Introduction : In natural product research, bioassay-guided fractionation was previously widely employed but is now judged to be inadequate in terms of time and cost, particularly if only known compounds are ultimately isolated. The development of metabolomics, along with improvements in analytical tools, allows comprehensive metabolite profiling. This enables dereplication to target unknown active compounds early in the purification workflow. Objectives :Starting from an ethanolic extract of violet leaves, this study aims to predict redox active compounds within a complex matrix through an untargeted metabolomics approach and correlation analysis. Methods : Rapid fractionation of crude extracts was carried out followed by multivariate data analysis (MVA) of liquid chromatography–high resolution mass spectrometry (LC–HRMS) profiles. In parallel, redox active properties were evaluated by the capacity of the molecules to reduce 2,2-diphenyl-1-picrylhydrazyl (DPPH·) and superoxide (O2 ·−) radicals using UV–Vis and electron spin resonance spectroscopies (ESR), respectively. A spectral similarity network (molecular networking) was used to highlight clusters involved in the observed redox activities. Results : Dereplication on Viola alba subsp. dehnhardtii highlighted a reproducible pool of redox active molecules. Polyphenols, particularly O-glycosylated coumarins and C-glycosylated flavonoids, were identified and de novo dereplicated through molecular networking. Confirmatory analyses were undertaken by thin layer chromatography (TLC)–DPPH–MS assays and nuclear magnetic resonance (NMR) spectra of the most active compounds. Conclusion : Our dereplication strategy allowed the screening of leaf extracts to highlight new biologically active metabolites in few steps with a limited amount of crude material and reduced time-consuming manipulations. This approach could be applied to any kind of natural extract for the study of various biological activities