Biomedical subjects

P Thérond

Publications and source records attributed to P Thérond.

At least 19 recordsLinked to original sources

PubMed2005

Antioxidant activity of melatonin and a pinoline derivative on linoleate model system.

This study aimed at investigating the in vitro protective effects of GWC22, a novel pinoline derivative [6-ethyl-1-(3-methoxyphenyl)-2-propyl-1,2,3,4-tetrahydro-beta-carboline] chlorhydrate, against radiation-induced oxidation of linoleate initiated by hydroxyl radicals ((*)OH). Using linoleate micelles (10(-2) m) as lipid model, two indexes of peroxidation have been measured, i.e. conjugated dienes and hydroperoxides. Similar determinations were performed with melatonin in order to compare the protective effects of the two compounds. It was observed that, the higher the concentration of GWC22 (or melatonin) (3 x 10(-5) to 10(-4) m), the stronger the antioxidant ability. In these in vitro assays, GWC22 showed a better antioxidant effect than melatonin for a given antioxidant concentration. A reaction scheme has been proposed to explain the inhibitory effect of an antioxidant via the propagating steps of the lipid peroxidation. Indeed, we have suggested that melatonin and GWC22 may compete with the fatty acid to scavenge lipid peroxyl radicals (LOO(*)). We have estimated a lower limit for the LOO(*) rate constant for GWC22 (>/=1.4 x 10(5)/m/s) and for melatonin (>/=2.8 x 10(4)/m/s) assuming that the k-value of the propagating step in linoleate (LOO(*) + linoleate) was 1.4 x 10(3)/m/s. The difference of reactivity between melatonin and GWC22 in this model system is assumed to be related to their relative lipophilicity.

Animals↗

PubMed2004

Liquid chromatographic/electrospray ionization mass spectrometric identification of the oxidation end-products of metformin in aqueous solutions.

Metformin is an antihyperglycemic drug that exhibits some antioxidant properties. HO*-induced oxidation of metformin was studied in aqueous solution, in both aerated and deaerated conditions. Gamma radiolysis of water was used to generate HO* free radicals, capable of initiating one-electron oxidation of metformin. Oxidation end-products were identified by direct infusion mass spectrometry (MS) and high-performance liquid chromatography/mass spectrometry (HPLC/MSn): for every product, structure elucidation was based on its mass (simple mass spectra confirmed by HPLC/MS). In addition, fragmentation spectra (MS2, MS3 and MS4) and the determination of deuterium-hydrogen exchange sites provided valuable information allowing the complete identification of some of the end-products. At low radiation dose, four products were identified as primary ones, since they result from the direct attack of HO* radicals on metformin. These primary oxidation end-products were identified respectively as hydroperoxide of metformin, covalent dimer of metformin, methylbiguanide and 2-amino-4-imino-5-methyl-1,3,5-triazine. At high radiation dose, seven other products were identified as secondary ones, resulting from the HO*-induced oxidation of the primary end-products. A reaction scheme was postulated for the interpretation of the results.

Antioxidants↗

PubMed2004

[Diabetes mellitus, oxidative stress and advanced glycation endproducts].

Chronic hyperglycemia in diabetes mellitus is an oxidative stress created by an imbalance of prooxidants over antioxidant defenses. The pathogenesis would involve several mechanisms including glucose autoxidation, protein glycation, the polyol pathway, and overproduction of superoxide radicals in mitochondria and via NAD(P)H oxidase. Glycemic equilibrium plays a very important role in the prooxidant/antioxidant balance. Macromolecules such as found in the extracellular matrix, lipoproteins, and deoxyribonucleic acid also constitute targets for free radicals in diabetes mellitus. This oxidative tress is involved in the pathophysiology of diabetes complications. The chronic hyperglycemic status also favors glycation reactions (irreversible glucose binding on protein amino groups), thereby leading to advanced glycation endproducts. Via their recognition by cell receptors, advanced glycation endproducts also participate in the development of oxidative stress and the inflammatory status. Involvement of oxidative stress and advanced glycation endproducts in diabetes complications is the basis of the development of adjunct therapies with antioxidant and/or anti)advanced glycation endproducts molecules.

Antioxidants↗

PubMed2004

Human sperm lipid content is modified after migration into human cervical mucus.

The effect of the female genital tract on sperm is not well known. To investigate the effect of cervical mucus on the lipid content of human sperm, we co-incubated sperm and mucus samples in vitro such that the sperm were able to swim in and out of the mucus samples. High performance liquid chromatography and UV detection were used to measure the lipid contents of the sperm and cervical mucus before and after migration. The concentrations of cholesterol, vitamin E, sphingomyelin, diacyls and plasmalogens in sperm were all approximately 45% lower after migration in cervical mucus and the cervical mucus was found to be enriched in some of these lipid species after the sperm migration. These results suggest that the cervical mucus selects a subpopulation of sperm with a lower lipid content. However, a concomitant efflux of various lipid classes from the sperm to the cervical mucus cannot be ruled out.

Cervix Mucus↗

PubMed2004

Radiation-induced peroxidation of small unilamellar vesicles of phosphatidylcholine generated by sonication.

The present study was aimed at determining the peroxidation of model membranes constituted of liposomes of 1-palmitoyl-2-linoleoyl-sn-glycero-3-phosphocholine (PLPC) submitted to hydroxyl free radicals (generated by gamma-radiolysis) attack. Liposomes of PLPC were prepared using the sonication technique, and dynamic light-scattering (DLS) measurements allowed characterization of the liposomal dispersions. Irradiation damages in sonication-generated liposomes were assessed by monitoring several oxidation products, such as conjugated dienes (by means of UV--visible spectrophotometry) and hydroperoxides (using reverse phase high-performance liquid chromatography (HPLC) associated with chemiluminescence detection). It has been shown that three different families of hydroperoxides are formed: the first one (at low radiation doses) results from HO. attack on the linoleyl chain of PLPC, giving phosphatidylcholine hydroperoxides possessing a conjugated dienic structure; the two others (at high radiation doses) are obtained by the secondary HO. attack on the primary hydroperoxide family. The quantification of these products associated with the comparison of their radiation-dose-dependent formation has provided valuable information concerning the mechanisms of their formation. Analysis by HPLC -- mass spectrometry has confirmed the presence of hydroperoxides and underlined various other products, like chain-shortened fragments and oxygenated derivatives of polyunsaturated sn-2 fatty acyl chain residues. Structural assignment proposals of some oxidation products have been proposed.

Chromatography, High Pressure Liquid↗

PubMed2001

Post-translational modifications of the regulatory subunits of cAMP-dependent protein kinases during G1/S progression.

During the G1/S transition of the cell cycle variations in the labelling by 8-N3-[32P]cAMP of the protein kinase A regulatory subunits RI and RII, used as a probe to monitor post-translational modifications that may regulate cAMP binding, were observed in synchronized HeLa cells. A decrease in 8-N3-[32P]cAMP labelling of RI, RII and RII phosphorylated by the catalytic subunit of PKA was correlated with the increased percentage of cells in phases G1. An increase in 8-N3-[32P]cAMP incorporated into the 54-kDa RII subunit during progression from G1 to S was correlated with an increase in intracellular cAMP. A transient increase in Mn-SOD activity was detected in cells arrested at the G1/S transition using two different techniques, suggesting that oxidative modulation of regulatory subunits by free radicals may modify cAMP binding sites during the cell cycle. Decreased photoaffinity labelling by 8-N3-[32P]cAMP of RI, RII and autophosphorylated RII subunits was found to be an inherent characteristic of PKA in the G1/S transition.

Azides↗

PubMed2001

Overexpression of copper zinc superoxide dismutase impairs human trophoblast cell fusion and differentiation.

The syncytiotrophoblast is the major component of the human placenta, involved in feto-maternal exchanges and secretion of pregnancy-specific hormones. Multinucleated syncytiotrophoblast arises from fusion of mononuclear cytotrophoblast cells. In trisomy 21-affected placentas, we recently have shown that there is a defect in syncytiotrophoblast formation and a decrease in the production of pregnancy-specific hormones. Due to the role of oxygen free radicals in trophoblast cell differentiation, we investigated the role of the key antioxidant enzyme, copper/zinc superoxide dismutase, encoded by chromosome 21 in in vitro trophoblast differentiation. We first observed that overexpression of superoxide dismutase in normal cytotrophoblasts impaired syncytiotrophoblast formation. This was associated with a significant decrease in mRNA transcript levels and secretion of hCG and other hormonal markers of syncytiotrophoblast. We confirmed abnormal cell fusion by overexpression of green fluorescence protein-tagged superoxide dismutase in cytotrophoblasts. In addition, a significant decrease in syncytin transcript levels was observed in superoxide dismutase-transfected cells. We then examined superoxide dismutase expression and activity in isolated trophoblast cells from trisomy 21-affected placentas. Superoxide dismutase mRNA expression (P < 0.05), protein levels (P < 0.01), and activity (P < 0.05) were significantly higher in trophoblast cells isolated from trisomy 21-affected placentas than in those from normal placentas. These results suggest that superoxide dismutase overexpression may directly impair trophoblast cell differentiation and fusion, and superoxide dismutase overexpression in Down's syndrome may be responsible at least in part for the failure of syncytiotrophoblast formation observed in trisomy 21-affected placentas.

Cell Differentiation↗

PubMed2001

Radiation induced peroxidation of polyunsaturated fatty acids: recent results on formation of hydroperoxides.

Aqueous solutions of linoleic acid were irradiated in air with gamma-rays of 137Cs. High pressure liquid chromatography (HPLC) was been used to separate and measure the production of hydroperoxides. The results obtained after reverse phase chromatography, associated with a microperoxydase for hydroperoxide detection, indicate the presence of two different hydroperoxides. One type of hydroperoxide was the major product obtained when the initial linoleic concentrations were below the critical micellar concentration (2 mM), and the second type was produced when the concentrations were above 2 mM. A further separation carried out on the second hydroperoxide by direct phase HPLC showed that it contains three compounds, mainly HPODE 9 and 13.

Chromatography, High Pressure Liquid↗

PubMed2000

The reaction of superoxide radical with N-acetylcysteine.

The interaction of superoxide radicals with N-acetylcysteine (RSH) in an aqueous solution of pH 7 using the technique of steady state radiolysis has been investigated in this paper. The radiolytic yield of the products (G value) of RSH consumption and disulfide of N-acetylcysteine (RSSR) formation has been determined. The G value of the products is not dependent on the concentration of RSH (at the plateau of dilution curve) or on the inverse of the square root of the dose rate (dose rate)(-1/2), from which it is concluded that in this reaction there is no character of chain reaction. The disulfide of N-acetylcysteine is the only sulfur final product. Hydrogen peroxide is not a reaction product, and accordingly the reaction of O(2)(*-) with RSH does not proceed via hydrogen atom abstraction from RSH. A reaction mechanism is proposed, and an overall rate constant of 68 M(-1) s(-1) has been estimated.

Acetylcysteine↗

PubMed2000

Increased oxidative damage to fibroblasts in skin with and without lesions in psoriasis.

Differences in oxidative damage, as measured by an increase in the carbonylation of macromolecules, were determined in situ with skin biopsies from psoriatic patients and controls. High levels of carbonyl residues were consistently detected in the dermis and never in the epidermis of sections of these skin biopsy samples. The dermis of psoriatic skin without lesions had a higher level of carbonylation than the dermis of normal skin. In this study, we found that there was more oxidative damage in cultured fibroblasts prepared from skin with and without lesions from psoriasis patients than in normal fibroblasts from the skin of age-matched controls. The extent of protein carbonylation in cell extracts was determined by immunoblotting, using an antidinitrophenylhydrazone antibody, and in intact cells was determined by immunocytochemical analysis with the same antibody. The higher level of carbonylation detected was used here as a measure of oxidative stress, and showed that some oxidative damage occurred before the appearance of typical psoriatic plaques. These results suggest that fibroblasts are affected before the onset of psoriasis and that this damage is independent of any inflammatory infiltrate.

Adult↗

PubMed2000

Modulation of copper/zinc superoxide dismutase expression and activity with in vitro differentiation of human villous cytotrophoblasts.

Due to the role of oxygen free radicals in trophoblast cell differentiation, we used the in vitro model of villous cytotrophoblast differentiation into the syncytiotrophoblast to investigate the modulation of the key antioxidant enzyme copper/zinc superoxide dismutase (SOD-1) in the human trophoblast during pregnancy. Cytotrophoblast cells were isolated from first-trimester and term placentae. SOD-1 mRNA levels were determined using real-time quantitative polymerase chain reaction, protein levels were determined by immunoblotting with a specific monoclonal antibody, and oxidoreductase activity was measured during syncytiotrophoblast formation in vitro. Interestingly, SOD-1 protein levels fell significantly (P< 0.001) during syncytiotrophoblast formation but no corresponding change in enzyme activity was observed. This apparent discrepancy may be related to different amounts of SOD-1 co-factor in the two cell types. Indeed the level of copper was significantly higher (P< 0.05) in syncytiotrophoblast as compared with cytotrophoblast. SOD-1 mRNA levels remained stable during cytotrophoblast differentiation. SOD-1 expression and activity were similar in cytotrophoblast cells isolated from first-trimester and term placentae, and in the differentiated syncytiotrophoblast in vitro. These results underline the need to determine SOD-1 protein expression and activity simultaneously in order to gain a better knowledge of its role in human trophoblast cell differentiation.

Cell Differentiation↗

PubMed2000

Biomarkers of oxidative stress: an analytical approach.

Oxidative stress is implicated in many pathological processes and results from a disruption of the prooxidant/antioxidant balance. This review will focus on noninvasive biomarkers of radical-induced damage in biological fluids and particularly in blood. Special attention will be addressed to new analytical methods for the measurement of radical-mediated alterations in the integrity of lipids, proteins and DNA.

Animals↗

PubMed2000

Evidence of the formation of different hydroperoxides in irradiated gamma-linolenate solutions: effect of micelle formation.

Peroxidation of unconjugated polyunsaturated fatty acids such as linolenic acid proceeds through a free radical chain mechanism and is accompanied by the formation of conjugated dienes such as hydroperoxides. In an investigation of radiation-induced oxidation of aqueous linolenate, we have measured two indexes of peroxidation: (1) conjugated dienes by means of absorption spectroscopy and (2) hydroperoxides by high-pressure liquid chromatography using detection of chemiluminescence. The experimental results indicate a strong effect of the concentration of linolenate on the yields of oxidized products. In addition, this work shows the quantitative production of two kinds of hydroperoxides. The ratio of these hydroperoxides is independent of the radiation dose but is dependent on the linolenate concentration. One hydroperoxide is formed predominantly below the critical micellar concentration (3 mM under our conditions), while the second is observed predominantly when micelles are formed in the aqueous medium. The influence of the composition of the medium on the nature of both hydroperoxides is discussed. [bj163]

Chromatography, High Pressure Liquid↗

PubMed1999

Major differences in oxysterol formation in human low density lipoproteins (LDLs) oxidized by OH/O2- free radicals or by copper.

The aim of our study was to determine the oxysterol formation in low density lipoproteins (LDLs) oxidized by defined oxygen free radicals (*OH/O2*-). This was compared to the oxysterol produced upon the classical copper oxidation procedure. The results showed a markedly lower formation of oxysterols induced by *OH/O2*- free radicals than by copper and thus suggested a poor ability of these radicals to initiate cholesterol oxidation in LDLs. Moreover, the molecular species of cholesteryl ester hydroperoxides produced by LDL copper oxidation seemed more labile than those formed upon *OH/O2*(-)-induced oxidation, probably due to their degradation by reaction with copper ions.

Cholesterol↗

PubMed1999

Inhibition of lecithin cholesterol acyltransferase by phosphatidylcholine hydroperoxides.

To gain insight into the nature of the lecithin-cholesterol acyltransferase inhibitory factor(s), we separated and collected the oxidation products from oxidized lipoproteins after lipoxygenase treatment. Isolated fractions identified by chemiluminescence, as hydroperoxides of phosphatidylcholine, were found to produce a significant reduction of lecithin-cholesterol acyltransferase activity. The reaction kinetics of lecithin-cholesterol acyltransferase with reconstitued high density lipoproteins were studied in the presence of 0.6 and 1.2 microM hydroperoxides of phosphatidylcholine. No significant changes in the apparent Vmax were observed but a concentration-dependent increase in slope of the reciprocal plots and in the apparent Km values was observed with increasing hydroperoxide concentrations. These results show that the active site of lecithin-cholesterol acyltransferase is not affected by the presence of phosphatidylcholine hydroperoxides. Nevertheless, hydroperoxides of phosphatidylcholine altered the reactivity of lecithin-cholesterol acyltransferase for reconstitued high density lipoproteins suggesting either an alteration of the binding of lecithin-cholesterol acyltransferase to the reconstitued high density lipoproteins or a competitive inhibition mechanism.

Apolipoprotein A-I↗

PubMed1999

Copper oxidation of in vitro dioleolylphosphatidylcholine-enriched high-density lipoproteins: physicochemical features and cholesterol effluxing capacity.

Susceptibility of lipoproteins to oxidation is partly determined by their content in endogenous antioxidants, but also by the polyunsaturated fatty acids (PUFA)/monounsaturated fatty acids (MUFA) ratio. The aim of our study was to enrich human high-density lipoproteins (HDLs) with dioleoylphosphatidylcholine (DOPC) in order to modify the PUFA/MUFA ratio while maintainig the alpha-tocopherol/PUFA ratio constant and to appreciate the consequences of this enrichment before and after copper-induced oxidation. The enrichment of HDLs with DOPC was obtained by incubation of these lipoproteins with DOPC liposomes and further reisolation of HDLs. The consequent 40% HDL enrichment in MUFA was concomitant with a 35% loss in PUFA (MUFA/PUFA ratio = 1.43). The enrichment of HDLs with DOPC led to a 40% decrease in alpha-tocopherol content, which kept a constant alpha-tocopherol/PUFA ratio. The DOPC-HDLs exhibited a lower oxidizability by copper than the nonenriched HDLs (NE-HDLs), as shown by their twofold longer lag phase and the threefold lower propagation rate. Moreover, DOPC-HDLs led to a six- to sevenfold lower production of hydroperoxide molecular species from phosphatidylcholine and cholesteryl esters than NE-HDLs after 24 h copper oxidation. With regard to the cholesterol effluxing capacity, copper oxidation of HDLs led to a decrease of this property. However, our results clearly showed that DOPC enrichment of HDLs allowed us to keep a better effluxing capacity than in NE-HDLs after 24 h oxidation (22.3% vs 17.4%, respectively). Since apo A-I was degraded as well in DOPC-HDLs as in NE-HDLs, the better effluxing capacity of DOPC-HDLs could not come from a preserved integrity of apo A-I. It could be partly related to the improved fluidity of oxidized DOPC-HDLs compared to oxidized NE-HDLs, as shown by electron spin resonance data (correlation-relaxation time at 24 degreesC = 2.20 ns vs 3.00 ns after 24 h oxidation, in DOPC-HDLs and in NE-HDLs, respectively). Besides, it could also be hypothesized that the sevenfold lower content of phosphatidylcholine hydroperoxides in DOPC-HDLs than in NE-HDLs after 24 h copper oxidation could be involved in the better ability of oxidized DOPC-HDLs to mobilize cellular cholesterol.

Apolipoprotein A-I↗

PubMed1999

Cholesteryl ester hydroperoxide lability is a key feature of the oxidative susceptibility of small, dense LDL.

Abundant evidence has been provided to substantiate the elevated cardiovascular risk associated with small, dense, low density lipoprotein (LDL) particles. The diminished resistance of dense LDL to oxidative stress in both normolipidemic and dyslipidemic subjects is established; nonetheless, the molecular basis of this phenomenon remains indeterminate. We have defined the primary molecular targets of lipid hydroperoxide formation in light, intermediate, and dense subclasses of LDL after copper-mediated oxidation and have compared the relative stabilities of the hydroperoxide derivatives of phospholipids and cholesteryl esters (CEs) as a function of the time course of oxidation. LDL subclasses (LDL1 through LDL5) were isolated from normolipidemic plasma by isopycnic density gradient ultracentrifugation, and their content of polyunsaturated molecular species of phosphatidylcholine (PC) and CE and of lipophilic antioxidants was quantified by reverse-phase high-performance liquid chromatography. The molar ratio of the particle content of polyunsaturated CE and PC species containing linoleate or arachidonate relative to alpha-tocopherol or beta-carotene did not differ significantly between LDL subspecies. Nonetheless, dense LDL contained significantly less polyunsaturated CE species (400 mol per particle) compared with LDL1 through LDL4 (range, approximately 680 to 490 mol per particle). Although the formation of PC-derived hydroperoxides did not vary significantly between LDL subspecies as a function of the time course of copper-mediated oxidation, the abundance of the C18:2 and C20:4 CE hydroperoxides was uniquely deficient in dense LDL (23 and 0.6 mol per particle, respectively, in LDL5; 47 to 58 and 1.9 to 2.3 mol per particle, respectively, in other LDL subclasses) at propagation half-time. When expressed as a lability ratio (mol hydroperoxides formed relative to each 100 mol of substrate consumed) at half-time, the oxidative lability of CE hydroperoxides in dense LDL was significantly elevated (lability ratio <25:100) relative to that in lighter, larger LDL particle subclasses (lability ratio >40:100) throughout the oxidative time course. We conclude that the elevated lability of CE hydroperoxides in dense LDL underlies the diminished oxidative resistance of these particles. Moreover, this phenomenon appears to result not only from the significantly elevated PC to free cholesterol ratio (1.54:1) in dense LDL particles (1.15:1 to 1.25:1 for other LDL subclasses) but also from their unique structural features, including a distinct apoB100 conformation, which may facilitate covalent bond formation between oxidized CE and apoB100.

Antioxidants↗

PubMed1999

High density lipoproteins (HDL) and the oxidative hypothesis of atherosclerosis.

The oxidative hypothesis of atherosclerosis classically implies a central role for low density lipoprotein (LDL) oxidation. However, new antiatherogenic properties have been recognized for high density lipoproteins (HDL), apart from their ability to reverse cholesterol transport. Indeed, native HDL could protect LDL from oxidation, thereby minimizing the deleterious consequences of this process. Several mechanisms have been suggested to explain this protective role. Two HDL-associated enzymes, paraoxonase and PAF-acetylhydrolase, detoxify oxidized phospholipids produced by lipid peroxidation. In addition, HDL could reduce hydroperoxides to their corresponding hydroxides. It has also been suggested that HDL could inhibit oxidized LDL-induced transduction signals. However, in vivo HDL oxidation in the subendothelial space would favor the atherosclerotic process. Indeed, atherogenic properties of these oxidized HDL partly result from some loss of their cholesterol effluxing capacity and from an inactivation of the lecithin-cholesterol acyltransferase, which is a HDL-associated enzyme involved in reverse cholesterol transport. Finally, oxidized HDL could induce cholesterol accumulation in macrophages. Further in-depth investigation is needed to assess these antagonistic effects and their consequences for the atherosclerotic process.

Arteriosclerosis↗