Monomerization of photosensitizers by ultrasound irradiation in surfactant micellar solutions.

The absorption and fluorescence properties of pheophorbide-a, Sodium salt of pheophorbide-a and its long chain (C20H39) ester (Pheophytine) were investigated in air-saturated micellar aqueous solutions before and after ultrasound irradiation (48 kHz, 10 min). The absorption spectra changed depending on the surfactant; cetyltrimethyl ammonium bromide (CTAB) or sodium dodecyl sulfate concentrations. The formation of different molecular species in various micellar solutions was estimated from the analysis of the absorption spectra. The absorption bands resulted from an aggregated form of the chromophore present in 50 mM phosphate buffer and in pre-micellar solutions. The specific bands of the aggregate disappeared with a simultaneous increase of the bands of monomer in normal micellar solution. The fluorescence spectra, the lifetimes and the fraction of each component (with a characteristic lifetime) of the chromophore in the micellar solutions changed significantly before and after ultrasound irradiation although the changes in absorption spectra were small. The fluorescence emission band at 710 nm due to the aggregate almost disappeared in the pre-micellar solution after ultrasound irradiation. The fraction of the short-lifetime component estimated for the aggregates decreased 55% in H2O or 85% in 2 mM CTAB, however the long-lifetime components increased after the ultrasound treatment. From these fluorescence properties, it was concluded that the aggregated molecules were converted to a stable monomeric form by ultrasound. Extrapolation of these data to in vivo situations suggests that pretreatment of certain photosensitizers with ultrasound in micellar solutions may lead to increased efficiency of photodynamic therapy since only the monomers are photodynamically active.

Free radical intermediates in sonodynamic therapy.

Current understanding of the mechanism of sonodynamic action (i.e. the ultrasound-dependent enhancement of the cytotoxic action of certain drugs--sonosensitizers) with potential applications for cancer therapy is presented. The experimental evidence suggests that sonosensitization is due to the chemical activation of sonosensitizers inside or in the close vicinity of hot collapsing cavitation bubbles to form sensitizer-derived free radicals either by direct pyrolysis or due to reactions with .H and .OH radicals, formed by pyrolysis of water. These free radicals (mostly carbon-centered) react with oxygen to form peroxyl and alkoxyl radicals. Unlike .OH and .H, which are also formed by pyrolysis inside cavitation bubbles, the reactivity of alkoxyl and peroxyl radicals with organic components dissolved in biological media is lower and hence have higher probability of reaching critical cellular sites. Sonodynamic therapy appears to be a promising modality for cancer treatment since ultrasound can penetrate deep within the tissue and can be focused in a small region of tumor to chemically activate relatively non-toxic molecules (e.g. porphyrins) thus minimizing undesirable side effects.

EPR spectroscopy of free radical intermediates of antiarrhythmic-antihypoxic drug stobadine, a pyridoindole derivative.

Mechanisms of antioxidant action of stobadine, a pyridoindole derivative with cardioprotective and antihypoxic properties, has been probed using EPR spectroscopy. Oxidation of stobadine by PbO2/tBuOOH in benzene results in the formation of nitroxide radical observable directly by EPR spectroscopy at room temperature, indicating conversion of indolic amino group to the corresponding nitroxide.

EPR characterization of free radical intermediates formed during ultrasound exposure of cell culture media.

Free radicals and/or hydrogen peroxide produced by exposure of cells to ultrasound are potentially cytotoxic and mutagenic. The formation and type of free radical species can be substantially modulated by the chemical composition of the media in which the ultrasound exposures of cells are carried out. In the current study, we examined the free radical intermediates formed during ultrasound exposure of a typical cell culture medium (RPMI-1640); the dominant free radicals that were identified by spin trapping were derived from the hydrophobic amino acids Trp, Leu, and Phe, and were formed by hydrogen abstraction from these amino acids. Compared to exposures in phosphate-buffered saline, the yield of *OH radicals and H2O2 was significantly reduced in the cell culture medium, glucose (the main organic component in the medium), and the hydrophobic amino acids (Trp, Phe, Tyr, Leu, Val, Met) being chiefly responsible for this effect. In contrast, other nonhydrophobic amino acids did not contribute significantly to the *OH or H2O2 decrease. These findings are consistent with the accumulation of hydrophobic solutes at the liquid-gas interface of the collapsing cavitation bubbles resulting in increased efficiency of radical scavenging.

Effects of cysteamine and cystamine on the sonochemical accumulation of hydrogen peroxide--implications for their mechanisms of action in ultrasound-exposed cells.

Based on the observed cytoprotective effect of the intracellularly permeable radical scavenger cysteamine (+NH3CH2CH2SH) in cells exposed to ultrasound and the lack of protection by its oxidized cell-nonpermeable form, cystamine (+NH3CH2CH2S-SCH2CH2NH3+), it was suggested that inertial cavitation (the growth of small gas bubbles present in the liquid exposed to ultrasound and their subsequent violent collapse) and associated free radical production may occur intracellularly (Radiat. Res. 89:369; 1982). Here we demonstrate that high concentrations (> 10 mM) of the thiol cysteamine effectively lower H2O2 yields following ultrasound exposure in argon- and air-saturated phosphate buffered saline (PBS), while cystamine is less effective under argon and practically without effect in air-saturated PBS. Direct removal of H2O2 by cysteamine is the dominant mechanism while scavenging of the H2O2 precursors .OH and superoxide plays a lesser role. Since H2O2 is a known cytotoxic species capable of penetrating cells if produced extracellularly, these results offer an alternative hypothesis for the protective effect of cysteamine and the lack of protection by cystamine, based on their differential ability to lower ultrasound-dependent H2O2 yields, without the necessity of invoking intracellular cavitation.

Effect of gas-containing microspheres and echo contrast agents on free radical formation by ultrasound.

Stabilized microbubbles (microspheres) are widely used to enhance the contrast of ultrasound imaging. Our data provide direct evidence that the contrast agents, Levovist, PVC-AN (polyvinylidene chloride-acrylonitryl copolymer), and Albunex (compared to 5% human albumin), at concentrations comparable to those used for ultrasound imaging, enhance H2O2 production (through the superoxide-dependent pathway) in air-saturated aqueous solutions exposed to 47 kHz ultrasound above the cavitation threshold. These agents also act as scavengers of .H atoms and .OH radicals, thus lowering H2O2 formation (by recombination of .OH radicals) in argon-saturated solutions. EPR spin trapping also reveals that secondary radicals derived from the contrast agents are produced by reactions with .H and .OH which are formed by pyrolysis of water inside cavitation bubbles. In addition, the contrast agents themselves undergo pyrolysis reactions in the cavitation bubbles as demonstrated by formation of methyl radicals. Possible deleterious consequences of the formation of sonochemical intermediates may have to be assessed, particularly since some of the echo contrast agents have been shown to lower the cavitation threshold of diagnostic ultrasound. Unlike the microspheres formed from organic molecules, inorganic microspheres, Eccospheres, because of their stability and inert nature with respect to participation in free radical processes, appear to be suitable tools for enhancing the yields of aqueous sonochemical reactions.

Antioxidant and pro-oxidant effects of epinephrine and isoprenaline on peroxidation of LDL and lipid liposomes.

Antioxidant or pro-oxidant properties of epinephrine (EPI) and isoprenaline (ISO) were studied in the absence and presence of Fe2+, Fe3+ and Cu2+ ions. EPI and ISO (>2 micromol/l) inhibited peroxidation of low density lipoprotein (LDL) induced by 2, 2'-azobis(2-amidino-propane) (AAPH). EPI had a similar inhibitory potency as ISO, but their potency was several times higher than the potency of alpha-tocopherol (alpha-TOC). When the LDL peroxidation was induced by 5 micromol/l CuSO4, EPI and ISO enhanced LDL peroxidation at low concentrations (10micromol/l) and decreased peroxidation at higher concentrations (30 micromol/l). The compounds had a similar tendency to inhibit the peroxidation of phosphatidylcholine liposomes. EPI (3-30 micromol/l) inhibited lipid peroxidation of phosphatidylcholine liposomes induced by 2 mmol/l of AAPH, but it was less effective and even increased the peroxidation, when the samples contained 2 mmol/l AAPH with 50 micromol/l FeSO4 or 2 mmol/l AAPH with 20 micromol/l FeCl3. Inhibition of lipid peroxidation by EPI was also observed when studying decreased oxygen consumption, when the peroxidation of linoleic acid was induced by lipoxidase. In conclusion, EPI and ISO reduced lipid peroxidation, but they exhibit pro-oxidant properties in the presence of Fe2+, Fe3+ or Cu2+ ions, depending on the catecholamine and ionic concentration.

Nitric oxide formation from hydroxylamine by myoglobin and hydrogen peroxide.

Hydroxylamine (HA), which is a natural product of mammalian cells, has been shown to possess vasodilatory properties in several model systems. In this study, HA and methyl-substituted hydroxylamines, N-methylhydroxylamine (NMHA) and N,N-dimethylhydroxylamine (NDMHA), have been tested for their ability to generate free diffusible nitric oxide (NO) in the presence of myoglobin (Mb) and hydrogen peroxide. A NO-specific conversion of 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide (carboxy-PTIO) to 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl (carboxy-PTI), measured by electron spin resonance (ESR) spectroscopy, along with nitrite and nitrate production, was observed for HA but not for NMHA and NDMHA. ESR measurements at 77 K showed the formation of the ferrous nitrosyl myoglobin, Mb-NO, in the reaction mixtures containing Mb, H2O2 and HA. Our data also demonstrate that Mb-NO is an end product of the reaction pathway involving Mb, H2O2 and HA, rather than a reaction intermediate in the formation of NO. In summary, our results demonstrate a possible pathway of NO formation from HA, however, the significance of this mechanism for bioactivation of HA in vivo is unknown at the present time.

Sonodynamic toxicity of gallium-porphyrin analogue ATX-70 in human leukemia cells.

Low concentrations (> or = 1 microM) of the gallium-porphyrin analogue ATX-70 significantly enhanced cellular toxicity in human leukemia HL-525 cells exposed to 50 kHz ultrasound. The mechanism of this ATX-70-dependent sonosensitization is unknown, but we have established the requirement of extracellular localization of ATX-70 molecules for sonosensitization. Short-lived toxic intermediates produced from ATX-70 by ultrasound are implicated in the mechanism, since no cytotoxicity was found when medium containing ATX-70 was sonicated and subsequently added to the cells. However, we were unable to demonstrate the existence of radical intermediates by EPR spin trapping with the nitroso spin trap, DBNBS, and ATX-70-dependent sonotoxicity could not be ameliorated by the addition of up to 70 mM POBN and DMPO spin traps during ultrasound exposure.

Inhibition of rat and human cytochrome P4502E1 catalytic activity and reactive oxygen radical formation by nitric oxide.

Nitric oxide (NO) reacts with heme-containing enzymes, including certain isoforms of cytochrome P450. Cytochrome P4502E1 (CYP2E1) is induced by ethanol and plays an important role in the toxicity of ethanol and other hepatotoxins. CYP2E1 is also very effective in generating reactive oxygen intermediates such as superoxide radical and H2O2, oxidizing ethanol to the 1-hydroxyethyl radical, and has a high NADPH oxidase activity. The effect of NO on CYP2E1 catalytic activity and generation of reactive oxygen intermediates was evaluated. Incubating liver microsomes isolated from rats treated with pyrazole to induce high levels of CYP2E1, with gaseous NO or NO released from a variety of NO donors such as SNAP, DEA/NO, spermine/NO, and GSNO, resulted in a loss of CYP2E1 catalytic activity with specific substrates such as p-nitrophenol or dimethylnitrosamine. Trapping of NO with hemoglobin resulted in protection of CYP2E1 activity against the inactivation by NO. There was no effect by analogues of the donors which do not release NO nor was there any effect by NO on NADPH-cytochrome P450 reductase activity. Inactivation of CYP2E1 by NO was not prevented by superoxide dismutase or catalase, suggesting that superoxide, H2O2, or peroxynitrite were not responsible for the actions of NO. The inactivated CYP2E1 was not degraded nor did it lose its epitope sites as shown by Western blot analysis. Associated with loss of CYP2E1 catalytic activity was a decrease in the formation of superoxide radical and H2O2, in microsomal lipid peroxidation catalyzed by low, but not high concentration of iron, and in consumption of NADPH. Oxidation of ethanol to the 1-hydroxyethyl radical was also inhibited by NO. ESR experiments indicated the formation of stable heme-NO complexes with CYP2E1. NO appears to compete with O2 and CO for binding to CYP2E1 as incubation with gaseous NO, or NO donors inhibited formation of the characteristic CO binding spectrum of P450. Microsomes isolated from a stably transfected HepG2 cell line expressing only CYP2E1 were also inactivated by NO, validating interaction of NO with this isoform of P450. These results indicate that NO inhibits CYP2E1 catalytic activity and generation of reactive radical intermediates. NO may prevent toxicity of agents which require bioactivation by P450 isoforms such as CYP2E1 and in generation of reactive intermediates by CYP2E1.

Lipoxygenase inhibition and antioxidant properties of bisbenzylisoqunoline alkaloids isolated from Mahonia aquifolium.

Products of lipoxygenase metabolism are known to play a role in the pathogenesis of psoriasis. Six bisbenzylisoquinoline (BBIQ) alkaloids, oxyacanthine, armoline, baluchistine, berbamine, obamegine, aquifoline, isolated from Mahonia aquifolium, were tested for lipoxygenase inhibition. Berbamine and oxyacanthine were the most potent lipoxygenase inhibitors, whereas aromoline and baluchistine exhibited only very low potencies. Oxyacanthine and berbamine were also among the most active compounds to inhibit lipid peroxidation. Between the results of lipoxygenase inhibition and the lipid peroxidation a linear correlation was found (r = 0.9533). Our data suggest that in the mechanism of lipoxygenase inhibition by these alkaloids, inhibition of lipid peroxide substrate accumulation, either by direct reaction with peroxide or by scavenging or lipid-derived radicals, may play a role. Inhibition of lipoxygenase by these compounds may contribute to the therapeutic effect of Mahonia aquifolium extracts in treatment of diseases in pathogenesis of which he products of lipoxygenase metabolism are involved.

EPR spin trapping study of the decomposition of azo compounds in aqueous solutions by ultrasound: potential for use as sonodynamic sensitizers for cell killing.

Sonodynamic therapy, a promising new approach to cancer treatment, is based on synergistic cell killing by combination of certain drugs (sonosensitizers) and ultrasound. Although the mechanism of sonodynamic action is not understood, the role of free radicals produced from sonosensitizers by ultrasound is implicated. In this work, we studied formation of free radicals during the decomposition of several water-soluble azo compounds by 50 kHz ultrasound in aqueous solutions. Using the spin trap 3,5-dibromo-4-nitrosobenzene sulfonate (DBNBS) tertiary carbon-centered radicals from 2,2'-azobis (N,N'-dimethyleneisobutyramidine) dihydrochloride (VA-044), 2-(carbamoylazo)-isobutyronitrile (V-30), and 2,2'-azobis (2-amidinopropane) dihydrochloride (AAPH) and .CH3 radicals from 1,1'-azobis (N,N'-dimethylformamide) (ADMF) were detected in argon-saturated solutions and the corresponding oxygen-centered radicals (alkoxyl and peroxyl) from VA-044, V-30, and AAPH were identified using the spin trap 5,5'-dimethyl-1-pyrroline-N-oxide (DMPO) in aerated sonicated solutions. No free radicals from 4,4'-dihydroxyazobenzene-3,3'-dicarboxylic acid, disodium salt (DHAB) could be found in either system. While VA-044 and AAPH could also be readily decomposed by heat (42.5 degrees C and 80 degrees C), V-30 decomposition only occurred in the ultrasound-exposed solutions. The most likely mechanism of decomposition of azo compounds by ultrasound is their thermolysis in the heated shell of the liquid surrounding cavitating bubbles driven by ultrasound and/or by pyrolysis inside these bubbles. Experiments using scavengers of .OH and .H, which are produced by sonolysis in aqueous solutions, demonstrated that these radicals are not involved in the ultrasound-mediated radical production from the azo compounds. Due to the known cytotoxic potential of free radicals produced from azo compounds, the use of these compounds as ultrasound sensitizers appears to be a promising approach for sonodynamic cell killing.

Peroxyl radical formation in aqueous solutions of N,N-dimethylformamide, N-methylformamide, and dimethylsulfoxide by ultrasound: implications for sonosensitized cell killing.

Sonodynamic therapy, which refers to a synergistic effect of drugs and ultrasound, is a promising new modality for cancer treatment. The sonodynamic effect was found for a number of structurally unrelated compounds, and the underlying mechanisms are still unknown. Recently, Jeffers et al. (J. Acoust. Soc. Am. 97:669-676; 1995) have shown that the sonodynamic action of nontoxic concentrations of N,N-dimethylformamide (DMF), N-methyl formamide (MMF), and dimethylsulfoxide (DMSO) combined with ultrasound, on killing of cultured HL-60 human promyelocytic leukemia cells, and attributed this toxic effect to unknown short lived reactive species produced from these solutes by ultrasonic cavitation. Using the spin trap 3,5-dibromo-4-nitrosobenzene sulfonate (DBNBS) in nitrogen-saturated aqueous solutions of DMF, MMF, or DMSO exposed to 50 kHz ultrasound, we detected formation of .CH3 and .CH2N(CH3)CHO radical adducts for DMF, mostly .CH2NHCHO adducts for MMF, and .CH3 adducts for DMSO. These radicals were formed either by reactions of the solutes with ultrasound-generated .H and .OH radicals (such as .CH2R-type radicals in DMF and MMF, and .CH3 radicals in DMSO), or by direct pyrolysis of the weak bonds in the solute molecules (e.g., .CH3 radicals from DMF). In air-saturated sonicated solutions these carbon centered radicals were converted to the corresponding peroxyl radicals and spin trapped with 5,5-dimethyl-1-pyrroline-N-oxide (DMPO); .OOCH2N(CH3)CHO radicals were identified in DMF, .OOCH2NHCHO radicals in MMF, and .OOCH3 radicals in DMSO solutions. We suggest that these radical species by virtue of their longer lifetimes and higher selectivity, compared to .OH radicals, which are also formed in sonicated solutions, are the species responsible for sonodynamic cell killing by the combined effect of ultrasound with DMF, MMF, or DMSO.

Increased cytotoxicity of 3-morpholinosydnonimine to HepG2 cells in the presence of superoxide dismutase. Role of hydrogen peroxide and iron.

3-Morpholinosydnonimine (SIN-1) is widely used to generate nitric oxide (NO(x).) and superoxide radical (O2-.). The effect of SOD on the toxicity of SIN-1 is complex, depending on what is the ultimate species responsible for toxicity. SIN-1 (< 1 mM) was only slightly toxic to HepG2 cells. Copper, zinc superoxide dismutase (Cu,Zn-SOD) or manganese superoxide dismutase (Mn-SOD) increased the toxicity of SIN-1. Catalase abolished, while sodium azide potentiated, this toxicity, suggesting a key role for H2O2 in the overall mechanism. Depletion of GSH from the HepG2 cells also potentiated the toxicity of SIN-1 plus SOD. Although Me2SO, sodium formate, and mannitol had no protective effect, iron chelators, thiourea and urate protected the cells against the SIN-1 plus Cu,Zn-SOD-mediated cytotoxicity. The cytotoxic effect of Cu,Zn-SOD but not Mn-SOD, showed a biphasic dose response being most pronounced at lower concentrations (10-100 units/ml). In the presence of SIN-1, Mn-SOD increased accumulation of H2O2 in a concentration-dependent manner. In contrast, Cu,Zn-SOD increased H2O2 accumulation from SIN-1 at low but not high concentrations of the enzyme, suggesting that high concentrations of the Cu,Zn-SOD interacted with the H2O2. EPR spin trapping studies demonstrated the formation of hydroxyl radical from the decomposition of H2O2 by high concentrations of the Cu,Zn-SOD. The cytotoxic effect of the NO donors SNAP and DEA/NO was only slightly enhanced by SOD; catalase had no effect. Thus, the oxidants responsible for the toxicity of SIN-1 and SNAP or DEA/NO to HepG2 cells under these conditions are different, with H2O2 derived from O2-. dismutation playing a major role with SIN-1. These results suggest that the potentiation of SIN-1 toxicity by SOD is due to enhanced production of H2O2, followed by site-specific damage of critical cellular sites by a transition metal-catalyzed reaction. These results also emphasize that the role of SOD as a protectant against oxidant damage is complex and dependent, in part, on the subsequent fate and reactivity of the generated H2O2.

Effect of gallium-porphyrin analogue ATX-70 on nitroxide formation from a cyclic secondary amine by ultrasound: on the mechanism of sonodynamic activation.

Sonodynamic therapy is a promising new modality for cancer treatment based on the synergistic effect on tumor cell killing by combination of a drug (typically a photosensitizer) and ultrasound. The mechanism of sonodynamic action was suggested to involve photoexcitation of the sensitizer by sonoluminescent light, with subsequent formation of singlet oxygen. In this work we studied the aqueous sonochemical reactions of the gallium-porphyrin derivative ATX-70, one of the most active sonodynamic agents found, using 50 kHz ultrasound. The experiments were carried out in the presence of 2,2,6,6-tetramethyl-4-piperidone hydrochloride (TMP), which reacts with singlet oxygen or .OH radicals to give the EPR-detectable nitroxide 2,2,6,6-tetramethyl-4-piperidone-N-oxyl (TMP-NO). Recently it has been suggested that the enhancement of TMP-NO yields in the presence of aqueous solutions of ATX-70 exposed to ultrasound was evidence for the formation of singlet oxygen in the system. Our results show that the surfactant cetyltrimethylammonium bromide (CTAB) can mimic the ATX-70-induced increase in the TMP-NO signal, but it fails to reproduce the behavior of ATX-70 in D2O: while the yields of TMP-NO in the presence of ATX-70 increase in D2O, the opposite effect was found with the surfactant CTAB. However, our data show that the increased TMP-NO yields in D2O are paralleled by an increased concentration of ATX-70 dimer, a form that is inactive in the photochemical generation of singlet oxygen. Our finding that the ATX-70-dependent enhancement of the TMP-NO signal was highest at approximately 20% O2, in both N2/O2 and argon/O2 mixtures, and decreased with increasing oxygen concentration is not compatible with the singlet oxygen mechanism. Finally, our results on the temperature dependence of the ATX-70-induced formation of TMP-NO are not consistent with the photochemical excitation of ATX-70 by sonoluminescent light: the ATX-70-dependent enhancement of TMP-NO signal increased with temperature in the range 10-25 degrees C, while the intensity of sonoluminescence of aqueous solutions both in multiple-bubble fields and in single-bubble experiments is known to decrease with increasing temperature.

Lipoxygenase inhibition and antioxidant properties of protoberberine and aporphine alkaloids isolated from Mahonia aquifolium.

Products of lipoxygenase metabolism play a role in the pathogenesis of psoriasis. Four protoberberine alkaloids, berberine, oxyberberine, jatrorrhizine, columbamine, and two aporphine alkaloids, magnoflorine, and corytuberine, isolated from Mahonia aquifolium, were tested for lipoxygenase inhibition. Oxyberberine, corytuberine, and columbamine were the most potent lipoxygenase inhibitors tested, whereas berberine and magnoflorine exhibited only low potencies. A strong linear correlation (r = 0.866) between lipoxygenase inhibition and lipid antioxidant properties of these compounds was found. These data suggest that the mechanism of lipoxygenase inhibition by these alkaloids may be linked to the inhibition of lipid hydroperoxide substrate accumulation. Inhibition of lipoxygenase by these compounds may contribute to the therapeutic effect of M. aquifolium extracts in the treatment of psoriasis.

Redox intermediates of flavonoids and caffeic acid esters from propolis: an EPR spectroscopy and cyclic voltammetry study.

The redox properties of flavonoids: chrysin (1), tectochrysin (2), galangin (3), isalpinin (4), pinostrobin (5), pinobanksin (6), pinobanksin-3-acetate (7), and of caffeic acid ester (8) and diacetylcaffeic acid ester (9), all isolated from propolis, were investigated by cyclic voltammetry in acetonitrile. The choice of aprotic solvent lowered the reactivity of the radical intermediates and made possible to identify redox steps and intermediates not detected so far. The oxidation potentials (vs. saturated calomel electrode) of the investigated compounds were in the region of 1.5 V for 3 and 4; 1.9 V for 1, 2, and 5; 2.0 V for 6 and 7; 1.29 V for 8; and 2.3 V for 9. These oxidation potentials were mainly influenced by the presence of a double bond in 2,3-position and substituent R1 in position 3. Comparison with our earlier data revealed that flavonoids, 1-4, and caffeic acid ester 8 with lower oxidation potentials showed the maximal lipid antioxidant activity, whereas those with higher potentials (5, 6, 7, and 9) are less active. On reduction of 1-9 several one-electron-steps were typically observed in the potential regions: -1.5 V, -1.8 V, and -2 V. where in simultaneous EPR experiments anion radicals of 1 and 3 were observed with the center of unpaired spin density on ring A. Upon oxidation of flavonoids 1-4 carbonyl carbon-centered radicals, .C(O)R, were identified as consecutive products using the EPR spin trapping technique.

Superoxide scavenging by thiol/copper complex of captopril--an EPR spectroscopy study.

Scavenging of superoxide radical by angiotensin converting enzyme (ACE) inhibitor captopril (CAP), a thiol compound, was studied by several investigators and the results were contradictory; while some reported a high superoxide scavenging activity of CAP others found that CAP removed superoxide inefficiently. In this work we show that in the presence of copper ions the apparent rate of superoxide removal by CAP (molar ratio CAP:CuSO4 4:1) was two orders of magnitude higher (approximately 1.5 x 10(5) M-1s-1 at pH 7.4) than the literature value for superoxide scavenging by CAP alone (< 10(3) M-1s-1 at pH 7.4). We presume that in the presence of copper ions a CAP/copper complex with a SOD-mimicking activity is being formed. Similar results were also obtained with another thiol glutathione (GSH). The possible role of the CAP/copper complexes in the anti-inflammatory effect of CAP is discussed.

Phospholipid hydroperoxides are precursors of lipid alkoxyl radicals produced from anoxia/reoxygenated endothelial cells.

Endothelial cells have been shown to generate primary oxygen-centered free radicals (hydroxyl, superoxide anion) during post-anoxic reoxygenation, but little evidence is available concerning subsequent initiation of lipid peroxidative injury in this model. Electron spin resonance (ESR) spectroscopy with alpha-phenyl-N-tert-butylnitrone (PBN) spin trapping was used to monitor lipid peroxidation (LPO)-derived free radicals formed by cultured bovine aortic endothelial cell suspensions exposed (37 degrees C) to anoxia (A, 45 min, N2 gas) and reoxygenation (R, 15 min, 95% O2/5% CO2). In some studies, superoxide dismutase (SOD, 10 micrograms/ml) was introduced just prior to R to assess the effects of this primary free radical scavenger on LPO-derived free radical production. At various times, aliquots were removed and PBN was introduced to either the cell suspension aliquot (8 mM PBN final, 1 min), or to the corresponding cell-free filtrate (60 mM PBN final), prior to extraction with toluene and ESR spectroscopy. A LPO-derived alkoxyl radical adduct of PBN (PBN/RO., hyperfine splitting alpha N = 13.63 G and alpha H = 1.94-1.98 G) was observed during R using both trapping procedures, with maximal production at 4-5 min and a second minor peak at 10 min. SOD effectively reduced PBN/RO. production and improved viability of A/R cells. In parallel studies, lipid hydroperoxide production was assessed in lipid extracts of A/R cells by high-performance liquid chromatography. Their separation profiles revealed a peak of oxidized lipid occurring between phosphatidylethanolamine (PE) and phosphatidylcholine (PC) in samples taken at 4-5 min and 10 min of R. Resolubilizing cell lipid extracts in oxygen-free benzene containing cobalt (II) acetylacetonate and PBN led to alkoxyl radical production, but only in the oxidized lipid samples, confirming the presence of hydroperoxides. These results suggest that A/R leads to primary free radical induced-lipid peroxidative injury to endothelial cells, as indicated by alkoxyl radical production originating from oxidized membrane phospholipids.

Reactive radical intermediates formed from illuminated nifedipine.

Nifedipine, (1,4-dihydro-2,6,dimethyl-4-(2-nitrophenyl)-3, 5-pyridinedicarboxylic acid dimethyl ester) a calcium channel blocker widely used in treatment of hypertension, is strongly photolabile. This may represent a problem in patients taking nifedipine and in handling of nifedipine samples. Reactive radical intermediates were determined and characterized in the process of nifedipine illumination using EPR spectroscopy. On illumination of nifedipine by daylight or by a mercury lamp, a nitroxide radical, RIIL-NIFNO.X was observed (in the first step), in various solvents like benzene, cyclohexane, methanol, acetonitrile, dimethylsulphoxide, or aqueous suspensions of liposomes. RIIL-NIF represents the nifedipine skeleton centered with phenyl group, and X is an EPR silent substituent. The generation of RIIL-NIFNO.X is coupled with the formation of nitroso compound, RIIL-NIFNO, as characterized by UV-visible spectroscopy. In a further step, RIIL-NIFNO abstracts hydrogen from nifedipine skeleton under the formation of RIIL-NIFNO.H radical. In addition to this, in system containing RIIL-NIFNO and unsaturated lipids, nitroxide radicals RIIL-NIFNO.RLIPIDS are formed probably via a pseudo Diels-Alder mechanism (RLIPIDS represents lipidic skeleton). The unusually easy photochemical activation of nifedipine is probably stimulated by photosensitization of its nitro group interacting with suitably positioned hydrogen or carboxylic methyl ester group from the pyridinyl ring.