Mechanism of one-electron oxidation of metformin in aqueous solution.

Hydroxyl free radical-induced oxidation of metformin was studied in aqueous solution as a function of the pH. Hydroxyl free radicals were generated by gamma radiolysis of water and the oxidation end-products were quantified by high-performance liquid chromatography coupled to mass spectrometry (HPLC/MS), as a function of the radiation dose. This work is a joint experimental and theoretical (DFT) approach that has paved the way towards a comprehensive rationalization of the one-electron mechanisms of MTF oxidation, as a function of the pH.

[Physico-chemical aspects of reactive oxygen species]. / Aspects physico-chimiques des espèces réactives de l'oxygène.

Reactive Oxygen Species (ROS), namely hydroxyl (*OH) and superoxide (O2*-) free radicals and hydrogen peroxide (H2O2), are involved in all oxidative stress phenomena. These latter occur in numerous pathological disorders such as, for example, cardiovascular diseases, diabetes or neurodegenerative diseases. Knowledge of thermodynamic (reduction potentials) and kinetic (rate constants) properties of ROS allows to draw up a rigorous overview of the chemical reactivity of these species. Hydroxyl free radicals (*OH) are powerful oxidants (very toxic species) which attack all the biomolecular targets (DNA, proteins, lipids...), giving other free radicals localized on the targets. Superoxide free radicals (O2*-) have a more graduated reactivity, since they don't directly react with biological molecules. However, some toxicity would be attributed to them via secondary radicular reactions. Other free radicals (of peroxyl RO2* and alkoxyl RO* types), belonging also to the ROS family, contribute by enhancement to oxidative stress.

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.

Radical-induced oxidation of metformin.

Metformin (1,1-dimethylbiguanide) is an antihyperglycaemic drug used to normalize glucose concentrations in type 2 diabetes. Furthermore, antioxidant benefits have been reported in diabetic patients treated with metformin. This work was aimed at studying the scavenging capacity of this drug against reactive oxygen species (ROS) like *OH and (O2*-)-free radicals. ROS were produced by gamma radiolysis of water. The irradiated solutions of metformin were analyzed by UV/visible absorption spectrophotometry. It has been shown that hydroxyl free radicals react with metformin in a concentration-dependent way. The maximum scavenging activity was obtained for concentrations of metformin > or = 200 micromol.L(-1), under our experimental conditions. An estimated value of 10(7) L.mol(-1).s(-1) has been determined for the second order rate constant k(*OH + metformin). Superoxide free radicals and hydrogen peroxide do not initiate any oxidation on metformin in our in vitro experiments.

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.

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.

An intracellular modulation of free radical production could contribute to the beneficial effects of metformin towards oxidative stress.

Metformin (dimethylbiguanide) is an antihyperglycemic agent used in type 2 diabetes. Beyond its action on glycemic control, metformin exhibits other intrinsic effects that could play a role in prevention against diabetes complications. Some studies thus reported an improvement in the antioxidant status in patients treated with metformin. This might be in part related to its property to limit formation of advanced glycation end products (AGEs) and to decrease the overproduction of free radicals in diabetic subjects. The aim of this study was to investigate the in vitro ability of metformin to modulate the action of reactive oxygen species (ROS) generated either by water gamma radiolysis or by stimulated human leukocytes. Our results showed that metformin at pharmacologically relevant concentrations was in vitro able to scavenge hydroxyl ((.)OH) but not superoxide (O(.-)(2)) free radicals and that hydrogen peroxide did not react with metformin. Nevertheless, when polymorphonuclear cells (PMN) are stimulated by phorbol myristate acetate (PMA), or above all by formyl methionine leucyl phenylalanine (fMLP), a systematic (although nonsignificant) decrease of the ROS-induced chimiluminescence (CL) was observed. These results suggest that metformin could directly scavenge ROS or indirectly act by modulating the intracellular production of superoxide anion, of which NADPH oxidase constitutes the major source. This could contribute to the additional benefits of metformin, especially those related to the improvement in the cardiovascular outcomes in diabetes.

Comparison of the effects of O2*-/HO* free radical- and copper ions-oxidized LDL or lipoprotein(a) on the endothelial cell releases of tissue plasminogen activator and plasminogen activator inhibitor-1.

We investigated the effects of low density-lipoproteins (LDL) and lipoprotein(a) [Lp(a)] oxidized by O2*-/HO* free radicals generated by gamma radiolysis of water, on the release of tissue Plasminogen Activator (tPA) and of its main inhibitor Plaminogen Activator Inhibitor-1 (PAI-1) by human umbilical vein endothelial cells (HUVEC). These effects were compared to those of lipoproteins issued from the same preparations but oxidized by the classical copper ions procedure. The results showed that O2*-/HO* free radical oxidized LDL and Lp(a) led to a dramatic decrease of PAI-1 release but did not affect tPA release, whereas copper oxidation of lipoproteins resulted in an increase in PAI-1 release and a decrease in tPA release. Chemical analysis revealed that O2*-/HO* free radical oxidized lipoproteins exhibited very much lower levels of phosphatidylcholine hydroperoxides, lysophosphatidylcholine and oxysterols (7-ketocholesterol, 7beta-hydroxycholesterol, 5,6beta-epoxycholesterol) than copper oxidized LDL. Thus, the discordant effects of O2*-/HO* oxidized and copper oxidized LDL and Lp(a) on the endothelial releases of PAI-1 and tPA appeared to be due to qualitatively and/or quantitatively different formation of oxidized components by the two oxidation processes.

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.

[Production of superoxide radicals with pulse radiolysis of water with high linear energy transfer]. / Production de radicaux superoxydes par radiolyse pulsée de l'eau à transfert d'énergie linéique (TEL) élevé.

The radiolysis of water with heavy ions of high linear energy transfer (LET) (-dE/dx) is characterized, in deaerated medium, by the production of superoxide anions, the radiolytic yields of which increase with the LET. Radiobiological interest in such radical species comes from the oxidative stress which may be generated by their dismutation in O2 and H2O2 in anoxic medium (radiotherapy with heavy ions). A brief review of the measurements of superoxide free radicals in aqueous solution by indirect or direct methods is presented. Moreover, some experimental results obtained by pulse radiolysis with Ar18+ ions (TEL = 290 keV x microm(-1)), are described. The interpretation of the kinetics takes into account the superoxide absorbance and that of hydrogen peroxide, which is present at the millisecond time scale.

[Radiolytic oxidation of tamoxifen with the free radicals OH- and/or HO2-]. / Oxydation radiolytique du tamoxifène par les radicaux libres OH et (ou) HO2.

Tamoxifen is the most widely used antiestrogen in the treatment of breast cancer. In this work, we have studied its antioxidant properties. We have investigated the ability of tamoxifen to scavenge, in vitro, *OH and (or) HO2* free radicals that are produced by water radiolysis. Aqueous solutions of tamoxifen of concentrations ranging between 10(-5) and 2.5 x 10(-5) M have been irradiated (gamma 137Cs) in aerated acidic medium (H3PO4 10(-3) M or HCOOH 10(-1) M). The results show that tamoxifen reacts quantitatively with *OH free radicals but does not react with HO2* free radicals under our experimental conditions.

Antioxidant effect of ethanol toward in vitro peroxidation of human low-density lipoproteins initiated by oxygen free radicals.

This study was designed to evaluate the effect of ethanol on the peroxidation of human low-density lipoprotein (LDL) initiated by oxygen free radicals (O(2)(.-) and (.)OH in the absence of ethanol; O(2)(.-) and ethanol-derived peroxyl radicals, RO(2)(.), in the presence of ethanol) generated by gamma radiolysis. Initial radiolytic yields as determined by several markers of lipid peroxidation [i.e. decrease in endogenous antioxidants alpha-tocopherol and beta-carotene, formation of conjugated dienes and of thiobarbituric acid-reactive substances (TBARS)] were determined in 3 g liter(-1) LDLs (expressed as total LDL concentration) in the absence of ethanol or its presence at six different concentrations (0.42-17 x 10(-2) mol liter(-1)). Ethanol acted as an antioxidant by decreasing the rate of consumption of LDL endogenous antioxidants and the yields of formation of lipid peroxidation products, and by delaying the onset of the propagation phase for conjugated dienes and TBARS. With regard to the different markers studied, except for alpha-tocopherol and beta-carotene consumption, the effect of ethanol did not appear to be dependent on its concentration. Indeed, (.)OH were scavenged by ethanol at the lowest ethanol concentration (0.42 x 10(-2) mol liter(-1)), leading to RO(2)(.). These RO(2)(.) resulted in lower radiation-induced yields related to endogenous antioxidant consumption or to formation of lipid peroxidation products (for example, approximately 10% of RO(2)(.) oxidized LDLs from TBARS). Thus, under our in vitro conditions, ethanol behaved as an antioxidant when added to the LDL solutions. This should be taken into account in the reported antioxidant activity of wine. This is also of interest when lipophilic compounds have to be added as ethanolic solutions to LDLs to evaluate in vitro their antioxidant activity toward LDL peroxidation.

Protection of endogenous vitamin E and beta-carotene by aminoguanidine upon oxidation of human low-density lipoproteins by *OH/O(2)*-.

This study was designed to evaluate the antioxidant effect of aminoguanidine toward human low-density lipoproteins (LDLs) initiated by oxygenated free radicals (*OH/O(2)*-) generated by gamma radiolysis. Initial radiolytic yields related to the markers of lipid peroxidation [i.e. decrease in endogenous alpha-tocopherol and beta-carotene, formation of thiobarbituric acid-reactive substances (TBARS) and conjugated dienes] were determined in 3 g liter(-1) LDLs (expressed as total LDL concentration) in the absence and presence of 10 different concentrations of aminoguanidine (from 0.04 to 5 mmol liter(-1)). Fluorescence and relative electrophoretic mobility of oxidized LDLs were also studied as markers that indirectly reflect the attack of the protein moiety of LDLs (namely apolipoprotein B). Our data clearly showed the inhibitory effect of aminoguanidine on lipid peroxidation induced in LDLs by *OH/O(2)*- in a concentration-dependent manner. This effect probably resulted from a scavenging activity of aminoguanidine toward *OH. In contrast, aminoguanidine did not appear to react significantly with O(2)*-, which resulted in a poor residual lipid peroxidation. Our data led us to determine an optimum [aminoguanidine]/[LDL] ratio ranging from 250 to 500 to obtain the best in vitro protection of LDLs under our experimental conditions. It is also of great interest that aminoguanidine was able to protect endogenous alpha-tocopherol and beta-carotene of LDLs upon *OH/O(2)*(-)-induced oxidation.

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]

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.

Antioxidant effect of probucol on RO2*/O2(*-)-induced peroxidation of human low-density lipoproteins.

This study was designed to evaluate the antioxidant effect of probucol on peroxidation of low-density lipoproteins (LDLs) initiated by oxygenated free radicals (O2*-) and ethanol-derived peroxyl radicals (RO2*) generated by gamma radiolysis. Initial radiolytic yields related to the markers of lipid peroxidation [i.e. decrease in endogenous alpha-tocopherol, formation of thiobarbituric acid-reactive substances (TBARS) and conjugated dienes] were determined as a function of LDL concentration (1.5 and 3 g l(-1), expressed as total LDL) and in the absence or the presence of probucol at different concentrations (2.3 x 10(-6), 3.5 x 10(-6), 9 x 10(-6) and 20.5 x 10(-6) mol l(-1)). Our results showed that probucol was able to decrease not only the yields of TBARS and conjugated dienes but also the levels of these peroxidation products obtained at high doses (2500 Gy) compared to LDLs without probucol. Under our conditions, probucol displayed an optimal antioxidant effect for an initial concentration in LDLs equivalent to 15 probucol molecules per LDL particle, which corresponded to a pharmacologically relevant concentration of probucol. Moreover, our data showed that probucol was unable to react with RO2* and thus did not protect LDL vitamin E from free radical attack. In addition, the scavenging capacity of probucol on O2*- appeared to be very poor, and probucol more likely reacted with LDL intermediate radical products. Finally, a very significant steady-state level of probucol remained in LDLs at high doses (up to 2500 Gy), equivalent to at least 40% of the initial concentration of probucol. This addressed the question of a mechanism for regeneration of probucol in LDLs. Our results as a whole suggested that the antioxidant effect of probucol in vivo could not be explained by its scavenging capacity with regard to RO2*/O2*- free radicals.

Determination of the yield of radiation-induced peroxidation of sodium linoleate in aqueous monomeric and micellar solutions.

Peroxidation of polyunsaturated fatty acids such as linoleic acid in aqueous micellar solution proceeds through a free-radical chain mechanism and is accompanied by the formation of conjugated dienes, some in the form of hydroperoxides. In the course of an investigation of radiation-induced oxidation of aqueous sodium linoleate, we have measured three indexes of peroxidation-conjugated dienes, hydroperoxides and thiobarbituric acid-reactive substances-by means of absorption spectroscopy, high-pressure liquid chromatography and spectrofluorimetry, respectively. There are linear correlations between the amounts of conjugated dienes, hydroperoxides and thiobarbituric acid-reactive substances. The radiolytic yields have been determined from the radiation dose dependence of the three markers of peroxidation as a function of sodium linoleate concentration. The results obtained indicate a strong effect of the concentrations of oxygen and linoleate on the yields of the products. The yields at different lipid concentrations display a large increase in chain propagation length; this is discussed in terms of the effect of micellar size.

Direct observation of HO2/O2- free radicals generated in water by a high-linear energy transfer pulsed heavy-ion beam.

The formation and decay of HO2/O2- radical from the radiolysis of water by heavy 36S16+ ions (2.7 GeV) have been observed by time-resolved absorption spectroscopy at 260 nm. The experiment was performed at the Grand Accélérateur National d'Ions Lourds (Caen, France). In deaerated water, for a linear energy transfer (LET) of 250 eV/nm, the yield of HO2/O2- is (6 +/- 2) x 10(-9) mol J-1. In aerated solution, an additional formation of O2- is observed due to the reaction of hydrogen atom and e(aq)- with oxygen. The experimental G values are compared to those obtained with light ions for the same LET. The importance of the initial velocity is discussed briefly.

Native and gamma radiolysis-oxidized lipoprotein(a) increase the adhesiveness of rabbit aortic endothelium.

Accumulation of monocyte-derived foam cells in the arterial intima is a major event in the development of atherogenesis. We have examined whether native and oxidized lipoprotein(a) (Lp(a)) can induce adhesion of monocytic cells to aortic endothelium. The extensive oxidation of paired samples of Lp(a) and low-density lipoprotein (LDL) was achieved by O2.-/OH. free radicals produced by gamma radiolysis of water, leading to similar values for the formation of peroxidation markers (conjugated dienes, TBARS, 8-epi-PGF2alpha) for both Lp(a) and LDL. Rabbit aortic segments were incubated for 5 h in the presence of equimolar concentrations of native and oxidized preparations of Lp(a) and LDL (125 micromol cholesterol/l, corresponding to 40 and 30 mg protein/l for Lp(a) and LDL, respectively). The aortic segments were incubated with rhodamin-isothiocyanate labeled U937 monocytic cells for 30 min and cell adhesion was quantified by fluorescent microscopy. Native Lp(a), and to a larger extent oxidized Lp(a), significantly increased U937 cell adhesion by 2.3 and 2.7 fold compared to controls (P < 0.005 and P < 0.001, respectively). Monocytic cell adhesion was also increased by native LDL (1.6 fold, P < 0.005), and to a greater extent by oxidized LDL (2.3 fold, P < 0.001). Thus native Lp(a) enhances the adhesive properties of the arterial endothelium which may account for its proatherogenic action. Furthermore, our results show that oxidized Lp(a), as well as oxidized LDL, are potent stimuli of monocyte adhesion to endothelial cells.