Jean-Pierre Souchard

Detection of superoxide anion released extracellularly by endothelial cells using cytochrome c reduction, ESR, fluorescence and lucigenin-enhanced chemiluminescence techniques.

Endothelium produces oxygen-derived free radicals (nitric oxide, NO&z.rad;; superoxide anion, O(2)(*-)) which play a major role in physiology and pathology of the vessel wall. However, little is known about endothelium-derived O(2)(*-) production, particularly due to the difficulty in assessing O(2)(*-) when its production is low and to controversies recently raised about the use of lucigenin-enhanced chemiluminescence. We compared four techniques of O(2)(*-) assessment when its production is low. In the present study, we have compared ferricytochrome c reduction, electron spin resonance (ESR) spectroscopy using DMPO as spin trap, hydroethidine fluorescence, and lucigenin-enhanced chemiluminescence to assess O(2)(*-) production in cultured bovine aortic endothelial cells (BAEC). We focused our study on extracellular O(2)(*-) production because the specificity of the signal is provided by the use of superoxide dismutase, and this control cannot be obtained intracellularly. We found that the calcium ionophore A23187 dose-dependently stimulated O(2)(*-) production, with a good correlation between all four techniques. The signals evoked by postconfluent BAEC were increased 2- to 7-fold in comparison to just-confluent BAEC, according to the technique used. Ferricytochrome c 20 microm rather than at 100 microm appears more suitable to detect O(2)(*-). However, in the presence of electron donors such as NADH or NADPH, lucigenin-enhanced chemiluminescence generated high amounts of O(2)(*-). Thus, ferricytochrome c reduction, electron spin resonance (ESR), and hydroethidine fluorescence appear as adequate tools for the detection of extracellular endothelium-derived O(2)(*-) production, whereas lucigenin may be artifactual, even when a low concentration of lucigenin is employed.

Electron spin resonance detection of extracellular superoxide anion released by cultured endothelial cells

OBJECTIVE AND METHODS Endothelium produces oxygen-derived free radicals which play a major role in vessel wall physiology and pathology. Whereas NO* production from endothelium has been extensively characterized, little is known about endothelium-derived O2-*. In the present study, we determined the O2-* production of bovine aortic endothelial cells (BAEC) using the spin trap 5,5-dimethyl-1 pyrroline-N-oxide (DMPO) and electron spin resonance (ESR) spectroscopy. RESULTS An ESR adduct DMPO-OH detected in the supernatant of BAEC after stimulation with the calcium ionophore A23187 originated from the trapping of extracellular O2-*, because coincubation with superoxide dismutase (30 U/ml) completely suppressed the ESR signal, whereas catalase (2000 U/ml) had no effect. A23187 stimulated extracellular O2-* production in a time- and dose-dependent manner. The coenzymes NADH and NADPH both increased the ESR signal, whereas a flavin antagonist, diphenylene iodonium, abolished the ESR signal. Phorbol myristate acetate potentiated, whereas bisindolylmaleimide I inhibited the A23187-stimulated O2-* production, suggesting the involvement of protein kinase C. These signals were not altered L-NAME, a NO-synthase inhibitor, suggesting that the endogenous production of NO* did not alter O2-* production. Finally, the amount of O2-* generated by A23187-stimulated post-confluent BAEC was one order of magnitude higher than that evoked by rat aortic smooth muscle cells stimulated under the same conditions.

Synthesis of estradiol–pheophorbide a conjugates: evidence of nuclear targeting, DNA damage and improved photodynamic activity in human breast cancer and vascular endothelial cells

The synthesis, physico-chemical properties, cellular localization and photocytotoxicity of estradiol-pheophorbide a conjugates in estrogen-dependent cancer and vascular endothelial cells are described with the aim of increasing the photodynamic activity by targeting the nucleus of both tumor and blood vessel cells.

Internalization of indium-labeled LDL through a lipid chelating anchor in human pancreatic-cancer cells as a potential radiopharmaceutical for tumor localization

Low‐density lipoproteins (LDL) labeled with indium via a lipid‐chelating agent, the bis(stearylamide) of diethylenetriaminepentaacetic acid (L), were evaluated as a potential radiopharmaceutical (111In‐L‐LDL) for tumor localization by studying their internalization in human pancreatic cancer cells (Capan‐1). Using Dil‐LDL (l,l′‐dioctadecyl‐3,3,3′,3′‐tetramethylindodicarbocyanine perchlorate‐LDL), this cell line was shown to bind human LDL with a high‐affinity saturable component and a low‐affinity non‐saturable (40%) component. The single saturable high‐affinity binding site had a KD of 27.5 ± 2.1 μg/ml and a maximal binding of 610 ± 7.5 ng/ml protein. Electron‐microscopic examination of the In‐L‐LDL particles revealed the peripheral distribution of the electron‐dense indium atoms at the outer surface of LDL. The modified LDL were then shown to be internalized by the cells. After conjugation of In‐L‐LDL to colloidal gold to follow the different stages of internalization, electron‐microscopic examination showed that the In‐L‐LDL gold conjugates were stuck to the external sheet of the plasma apical and microvilli membrane, into earlier and later endosomes and into multi‐vesicular bodies, suggesting the penetration of the In‐L‐LDL particles into lysosomal vacuoles. The observation of In‐L‐LDL‐gold conjugates in deep‐seated cytoplasm suggests that LDL could be employed as a drug‐transport vehicle for targeting cytotoxics or radionuclides close to the cell nucleus. Int. J. Cancer, 70:315–322, 1997.

2-substituted-3H-indol-3-one-1-oxides: preparation and radical trapping properties.

A series of 2-alkyl and 2-aryl substituted-3H-indol-3-one-1-oxides was prepared and evaluated for its radical trapping properties. Spin trapping and electron paramagnetic resonance experiments demonstrate the ability of these indolone-1-oxides to trap hetero- and carbon-centered radicals. The most stable spin adducts (lifetime of several hours) are obtained with 2-alkyl substituted nitrones, the 2-ethyl-5,6-dioxolo-3H-indolone-1-oxide, 5e and the 2-secbutyl-3H-indolone-1-oxide, 5f. These two nitrones are also sensitive to redox reactions in solution. Therefore this indolone-1-oxide series lacking a β-hydrogen atom gives rise to highly stable adducts with free radicals.

SYNTHESIS AND CHARACTERIZATION OF NEW AROMATIC THIONITRITES

Abstract Thionitrites (S-nitrosothiols) play an essential biological role as nitric oxide (NO.) carriers. Here, we present the synthesis. the characterization and the stability studies in solution of new aromatic thionitrites 1b-4b as potent nitric oxide donors. The four thionitrites were characterized by 1H NMR, UV-visible and IR spectroscopies. Their decomposition occurs within a few minutes in dichloromethane, and yields quantitatively the corresponding disulfide. NO. and the thiyl radicals coming from their decomposition were trapped by distinct spin traps to give characteristic EPR signals. 4b possesses the di-tert-butylphenol moiety responsible for the antioxidant properties of BHT and of the structurally-related drug probucol.

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.

EDTA and DTPA analogues of dipalmitoylphosphatidylethanolamine as lipopphilic chelating agents for metal labeling of LDL

Abstract Two lipophilic chelating agents (L) prepared by reaction of dipalmitoylphosphatidylethanolamine with the bis(anhydride) form of ethylenediaminetetraacetic acid (EDTA) or diethylenetriaminepentaacetic acid (DTPA) were characterized. L, indium-L or gadolinium-L complexes are soluble in buffered solutions and may be used for metal labeling of LDL.

SYNTHESIS OF NEW LIPOPHILIC ORTHO-DICARBORANES

Abstract The synthesis of several new lipoidal dicarborane compounds prepared by reaction of decaborane with several fatty mono- and bis-alkyl-1,3-diols is described.

Manganese(II) Complexes with Orotic Acid Derivatives as Scavengers of Superoxide Radicals

The Superoxide anion radical, O2 −, plays a key role in the initiation stage of oxidative damage in biological systems (1). The main line of defense in mammalian organisms for controlling intracellular, and to a lesser extent extracellular, O2 − radicals are the Cu/Zn and Mn-containing Superoxide dismutase (SOD) enzymes and, recently, the application of SOD as a drug has attracted much attention (2). Since various problems are associated with using an enzyme as a drug (cost, bioavailability, stability, immunogenicity), non-toxic and low-mass metal complexes that catalyze the dismutation of O2 − might be able to substitute for SOD in such applications.