Ange Mouithys-Mickalad

GENERATION OF SUPEROXIDE ANION BY MITOCHONDRIA AND IMPAIRMENT OF THEIR FUNCTIONS DURING ANOXIA AND REOXYGENATION IN VITRO

A small portion of the oxygen consumed by aerobic cells is converted to superoxide anion at the level of the mitochondrial respiratory chain. If produced in excess, this harmful radical is considered to impair cellular structures and functions. Damage at the level of mitochondria have been reported after ischemia and reperfusion of organs. However, the complexity of the in vivo system prevents from understanding and describing precise mechanisms and locations of mitochondrial impairment. An in vitro model of isolated-mitochondria anoxia-reoxygenation is used to investigate superoxide anion generation together with specific damage at the level of mitochondrial oxidative phosphorylation. Superoxide anion is detected by electron paramagnetic resonance spin trapping with POBN-ethanol. Mitochondrial respiratory parameters are calculated from oxygen consumption traces recorded with a Clark electrode. Respiring mitochondria produce superoxide anion in unstressed conditions, however, the production is raised during postanoxic reoxygenation. Several respiratory parameters are impaired after reoxygenation, as shown by decreases of phosphorylating and uncoupled respiration rates and of ADP/O ratio and by increase of resting respiration. Partial protection of mitochondrial function by POBN suggests that functional damage is related and secondary to superoxide anion production by the mitochondria in vitro.

Effects of propofol on endothelial cells subjected to a peroxynitrite donor (SIN-1)

We investigated the effect of propofol on endothelial cells subjected to the peroxynitrite (ONOO−) donor 3‐morpholino sydnonimine (SIN‐1). Cells were incubated overnight with 0.5, 1.0 or 2.0u2003mm SIN‐1, with or without 10−3u2003m propofol (Diprivan®). Cytotoxicity, assessed by measuring the release of pre‐incorporated 51Cr, increased when the concentration of SIN‐1 increased, and was significantly decreased by 10−3u2003m propofol (90%, 78% and 28% of protection against 0.5, 1.0 and 2.0u2003mm SIN‐1, respectively). Cell protection against 1u2003mm SIN‐1 was tested with 0.03–1.0u2003mm propofol and this was compared to tyrosine, a target molecule for peroxynitrite. Propofol protected cells in a dose‐dependent manner (ru2003=u20030.98; p <u200a0.001) and was as effective as tyrosine. Finally, using high‐performance liquid chromatography, we demonstrated that propofol reacted with ONOO− more rapidly than did tyrosine, inhibiting nitrotyrosine formation. In the absence of propofol, 3.5u2003mm ONOO− with 1u2003mm tyrosine yielded 39.6% nitrotyrosine, but nitrotyrosine was not produced when 5u2003mm propofol was added. We conclude that propofol protects endothelial cells against the toxicity of ONOO−. The anti‐oxidant properties of propofol can be partially attributed to its scavenging effect on peroxynitrite, a property that might be relevant in pathological situations involving a significant contribution of peroxynitrite to tissue damage.

EGb 761 protects liver mitochondria against injury induced by in vitro anoxia/reoxygenation

The present study investigated the protective effects of Ginkgo biloba extract (EGb 761) on rat liver mitochondrial damage induced by in vitro anoxia/reoxygenation. Anoxia/reoxygenation was known to impair respiratory activities and mitochondrial oxidative phosphorylation efficiency. ADP/O (2.57 +/- 0.11) decreased after anoxia/reoxygenation (1.75 +/- 0.09, p < .01), as well as state 3 and uncoupled respiration (-20%, p < .01), but state 4 respiration increased (p < .01). EGb 761 (50-200 microg/ml) had no effect on mitochondrial functions before anoxia, but had a specific dose-dependent protective effect after anoxia/reoxygenation. When mitochondria were incubated with 200 microg/ml EGb 761, they showed an increase in ADP/O (2.09 +/- 0.14, p < .05) and a decrease in state 4 respiration (-22%) after anoxia/reoxygenation. In EPR spin-trapping measurement, EGb 761 decreased the EPR signal of superoxide anion produced during reoxygenation. In conclusion, EGb 761 specially protects mitochondrial ATP synthesis against anoxia/reoxygenation injury by scavenging the superoxide anion generated by mitochondria.

Oxidation of tetrahydrobiopterin by peroxynitrite or oxoferryl species occurs by a radical pathway

The molecular mechanisms of tetrahydrobiopterin (BH4) oxidation by peroxynitrite (ONOO-) was studied using ultra-weak chemiluminescence, electron paramagnetic resonance (EPR) and UV-visible diodearray spectrophotometry, and compared to BH4 oxidation by oxoferryl species produced by the myoglobin/hydrogen peroxide (Mb/H2O2) system. The oxidation of BH4 by ONOO- produced a weak chemiluminescence, which was altered by addition of 50 mM of the spin trap α-(4-pyridyl-1-oxide)-N-tert butylnitrone (POBN). EPR spin trapping demonstrated that the reaction occurred at least in part by a radical pathway. A mixture of two spectra composed by an intense six-line spectrum and a fleeting weak nine-line one was observed when using ONOO-. Mb/H2O2 produced a short-living light emission that was suppressed by the addition of BH4. Simultaneous addition of POBN, BH4 and Mb/H2O2 produced the same six-line EPR spectrum, with a signal intensity depending on BH4 concentration. Spectrophotometric studies confirmed the rapid disappearance of the characteristic peak of ONOO- (302 nm) as well as substantial modifications of the initial BH4 spectrum with both oxidant systems. These data demonstrated that BH4 oxidation, either by ONOO- or by Mb/H2O2, occurred with the production of activated species and by radical pathways.

Metal-organic compounds: a new approach for drug discovery. N1-(4-methyl-2-pyridyl)-2,3,6-trimethoxybenzamide copper(II) complex as an inhibitor of human immunodeficiency virus 1 protease.

The use of metal-organic complexes is a potentially fruitful approach for the development of novel enzyme inhibitors. They hold the attractive promise of forming stronger attachments with the target by combining the co-ordination ability of metals with the unique stereoelectronic properties of the ligand. We demonstrated that this approach can be successfully used to inhibit the protease of the human immunodeficiency virus (type 1). Several ligands bearing substituents designed to interact with the catalytic site of the enzyme when complexed to Cu(2+) were synthesised. The inhibition pattern of the resulting copper(II) complexes was analysed. We showed that the copper(II) complex of N1-(4-methyl-2-pyridyl)-2,3,6-trimethoxybenzamide (C1) interacts with the active site of the enzyme leading to competitive inhibition. On the other hand, N2-pyridine-amide ligands and oxazinane carboxamide ligand were found to be poor chelators of the cupric ion under the enzymatic assay conditions. In these cases, the observed inhibition was attributed to released cupric ions which react with cysteine residues on the surface of the protease. While unchelated metal cations are not likely to be useful agents, metal chelates such as C1 should be considered as promising lead compounds for the development of targeted drugs.

High concentrations of histamine stimulate equine polymorphonuclear neutrophils to produce reactive oxygen species.

Abstract.Objective and Design: Because high concentrations of histamine are locally released in inflammation, we investigated the effects of supraphysiological doses of histamine on the production of reactive oxygen species (ROS) by neutrophils. ¶Materials and Methods: Isolated equine neutrophils were activated by 10-4 to 5 × 10-3 M histamine. The production of ROS and free radicals was estimated by luminol-enhanced chemiluminescence (CL) and electron spin resonance (ESR) with spin trapping technique. In this model of histamine-stimulated neutrophils, we tested the antagonists of H1 and H2 histamine receptors, the role of Ca2+ and Mg2+, the role of staurosporine and pertussis toxin (inhibitors of protein kinase C and proteins G) and the effects of superoxide dismutase, catalase, hydroxyl radical scavengers (phenylalanine and mannitol) and NG-monomethyl-L-arginine (L-NMMA), inhibitor of NO-synthase. ¶Results: Histamine (from 10-5 to 10-3 M) stimulated neutrophils to produce CL and ESR signals characterized by spin adducts of superoxide anion and/or hydroxyl radicals. The CL response was inhibited by 10-4 and 10-3 M H1 receptor antagonists (promethazine, pyrilamine, and diphenhydramine), by Ca2+ and Mg2+ depletion and by 10 nmoles staurosporine. CL was partially inhibited by pertussis toxin (4 μg/ mL). The ESR signals were practically suppressed by pyrilamine (an H1 receptor antagonist) and superoxide dismutase, and partially inhibited by catalase, hydroxyl radical scavengers and L-NMMA (respectively 59, ± 30% and 68% inhibition). ¶Conclusions: High concentrations of histamine stimulated the neutrophils to product ROS and free radicals via H1 receptors and the NADPH-oxidase pathway.

EPR studies of in vivo radical production by 3,3′,5,5′-tetrabromobisphenol A (TBBPA) in the Sprague–Dawley rat

Brominated flame retardants (BFRs) are present in many consumer products ranging from fabrics to plastics and electronics. Wide use of flame retardants can pose an environmental hazard and it is of interest to determine the mechanism of their toxicity. Of all the BFRs, 3,3,5,5-tetrabromobisphenol A (TBBPA) is produced in the largest volume. Previous studies by Szymanska et al. (2000) have shown that TBBPA is hepatotoxic in rats. We report here that when TBBPA (100 or 600 mg/kg) dissolved in DMSO and alpha-(4-pyridyl-1-oxide)-N-t-butylnitrone (POBN) was administered ip to male Sprague-Dawley rats the POBN/CH(3) spin adduct was detected by electron paramagnetic resonance (EPR) in the bile. When (13)C-DMSO was employed the POBN/C(13)H(3) adduct was observed. Also present in the bile was the 2,6-dibromobenzosemiquinone radical derived from 2,6-dibromohydroquinone, a known metabolite of TBBPA. Reaction of the 2,6-dibromobenzosemiquinone radical with oxygen would generate superoxide from which hydrogen peroxide can form by dismutation. The hydroxyl radical generated via the Fenton reaction from hydrogen peroxide reacts in vivo with DMSO to give the methyl radical which is trapped by POBN. These observations suggest that the hepatotoxicity of TBBPA in rats may be due to the in vivo generation of the hydroxyl radical as a result of redox reactions involving the TBBPA metabolite 2,6-dibromohydroquinone and its corresponding semiquinone radical.

In vitro evaluation of glutathione peroxidase (GPx)-like activity and antioxidant properties of some Ebselen analogues

Abstract Four analogues of Ebselen were synthesized and their glutathione peroxidase activity and antioxidant property evaluated and compared to Ebselen. Among the studied compounds, only diselenide [3] exhibited both glutathione peroxidase activity and radical-scavenging capability. Compounds [3] and [4] showed a strong inhibitory effect (53% and 43%, respectively) on the lipid peroxidation of linoleic acid compared to Ebselen and selenide derivatives ([1] and [2]) which were less active (28%, 26% and 18% inhibition, respectively). A concentration-dependent inhibitory effect was also found in the model of the formation of ABTS•+ radical cation: 65% and 89% inhibition for compound [3] at 10–4 M and 5 × 10–5 M, respectively, and 68% and 90% for compound [4], compared to 14% and 52% inhibition for Ebselen and the diselenides [1] and [2] (29%, 46% and 45%, 68%, respectively). By EPR spin trapping technique, the following inhibitory profile of the Ebselen analogues was observed towards the formation of thiyl radicals: Ebselen = [3]>[1]>[2]>[4]. Studies with compound [3] are in progress on oxidative stress cell models.

Oxygen consumption of equine articular chondrocytes: Influence of applied oxygen tension and glucose concentration during culture

We investigated the oxygen (O2) uptake of equine articular chondrocytes to assess their reactions to anoxia/re‐oxygenation. They were cultured under 5% or 21% gas phase O2 and at glucose concentrations of 0, 1.0 or 4.5 g/L in the culture medium (n = 3). Afterwards, the O2 consumption rate of the chondrocytes was monitored (oxymetry) before and after an anoxia period of 25 min. The glucose consumption and lactate release were measured at the end of the re‐oxygenation period. The chondrocytes showed a minimal O2 consumption rate, which was hardly changed by anoxia. Independently from the O2 tension, glucose uptake by the cells was about 30% of the available culture medium glucose, thus higher for cells at 4.5 g/L glucose (n= 3). Lactate release was also independent from O2 tension, but lower for cells at 4.5 g/L glucose (n= 3). Our observations indicated that O2 consumption by equine chondrocytes was very low despite a functional mitochondrial respiratory chain, and nearly insensitive to anoxia/re‐oxygenation. But the chondrocytes metabolism was modified by an excess of O2 and glucose.

ESR evidence for in vivo formation of free radicals in tissue of mice exposed to single-walled carbon nanotubes

Nanomaterials are being utilized in an increasing variety of manufactured goods. Because of their unique physicochemical, electrical, mechanical, and thermal properties, single-walled carbon nanotubes (SWCNTs) have found numerous applications in the electronics, aerospace, chemical, polymer, and pharmaceutical industries. Previously, we have reported that pharyngeal exposure of C57BL/6 mice to SWCNTs caused dose-dependent formation of granulomatous bronchial interstitial pneumonia, fibrosis, oxidative stress, acute inflammatory/cytokine responses, and a decrease in pulmonary function. In the current study, we used electron spin resonance (ESR) to directly assess whether exposure to respirable SWCNTs caused formation of free radicals in the lungs and in two distant organs, the heart and liver. Here we report that exposure to partially purified SWCNTs (HiPco technique, Carbon Nanotechnologies, Inc., Houston, TX, USA) resulted in the augmentation of oxidative stress as evidenced by ESR detection of α-(4-pyridyl-1-oxide)-N-tert-butylnitrone spin-trapped carbon-centered lipid-derived radicals recorded shortly after the treatment. This was accompanied by a significant depletion of antioxidants and elevated biomarkers of inflammation presented by recruitment of inflammatory cells and an increase in proinflammatory cytokines in the lungs, as well as development of multifocal granulomatous pneumonia, interstitial fibrosis, and suppressed pulmonary function. Moreover, pulmonary exposure to SWCNTs also caused the formation of carbon-centered lipid-derived radicals in the heart and liver at later time points (day 7 postexposure). Additionally, SWCNTs induced a significant accumulation of oxidatively modified proteins, increase in lipid peroxidation products, depletion of antioxidants, and inflammatory response in both the heart and the liver. Furthermore, the iron chelator deferoxamine noticeably reduced lung inflammation and oxidative stress, indicating an important role for metal-catalyzed species in lung injury caused by SWCNTs. Overall, we provide direct evidence that lipid-derived free radicals are a critical contributor to tissue damage induced by SWCNTs not only in the lungs, but also in distant organs.