Although oxygen radicals are thought to play a key role in the skin injury that is caused by protoporphyria, there is no direct evidence of generation of these radicals in vivo. This study measured the generation of oxygen radicals caused by visible light non-invasively in the skin of griseofulvin-induced protoporphyria model mice, using an in vivo electron spin resonance spectrometer equipped with a surface-coil-type resonator that could detect radicals within about 0.5 mm of the skin surface. A durable nitroxyl radical was administered intravenously as a probe. Light irradiation enhanced the decay of the nitroxyl signal in griseofulvin-treated mice, whereas light irradiation did not enhance the signal decay in control mice. The enhanced signal decay was completely suppressed by intravenous administration of hydroxyl radical scavengers, superoxide dismutase or catalase, or the intraperitoneal administration of desferrioxamine. The enhanced signal decay with illumination was reversible, and quickly responded to turning the light on and off. These observations suggest that the hydroxyl radical is generated via an iron-catalyzed reaction in the skin. This paper demonstrates, for the first time, the specific generation of oxygen radicals in response to light irradiation of the skin of protoporphyria model mice.

Lipid alkyl radicals generated from polyunsaturated fatty acids via chemical or enzymatic H-abstraction have been a pathologically important target to quantify. In the present study, we established a novel method for the quantification of lipid alkyl radicals via nitroxyl radical spintrapping. These labile lipid alkyl radicals were converted into nitroxyl radical-lipid alkyl radical adducts using 3-carbamoyl-2,2,5,5-tetramethyl-3-pyrroline-N -oxyl (Cm ⌬ P) (a partition coefficient between octanol and water is approximately 3) as a spin-trapping agent. The resulting Cm ⌬ P-lipid alkyl radical adducts were determined by HPLC with postcolumn online thermal decomposition, in which the adducts were degraded into nitroxyl radicals by heating at 100 ؇ C for 2 min. The resulting nitroxyl radicals were selectively and sensitively detected by electrochemical detection. With the present method, we, for the first time, determined the lipid alkyl radicals generated from linoleic acid, linolenic acid, and arachidonic acid via soybean lipoxygenase-1 or the radical initiator 2,2 -azobis(2,4-dimethyl-valeronitrile). Peroxidation of polyunsaturated fatty acids, which are components of cellular membrane and lipoproteins, leads to vital damage to several types of cells. Reactive oxygen/nitrogen species (1), such as hydroxyl radical, hypochlorous acid (2-4), peroxynitrite (5, 6), and singlet oxygen molecule (7-9) are involved in the peroxidation of polyunsaturated fatty acids in the inflammatory lesions. On the other hand, lipoxygenases are speculated to be involved in the peroxidation of polyunsaturated fatty acids in the atheromatous plaque (10). Commonly, lipid alkyl radicals seem to be necessarily produced as an intermediate in these reactions.To detect labile free radicals via ESR, spin-trapping techniques have been established, in which unstable radicals react with spin-trapping agents to form relatively stable radical adducts (11). To date, nitrone spin-trapping agents, including ␣ -[4-pyridyl-1-oxide] N -tert-butyl nitrone and 5,5 Ј -dimethyl-1-pyroline-N -oxide, have been used to detect these lipid-derived radicals (12-17). Furthermore, Mason and coworkers (13-15) established an HPLC/ESR system to identify the resulting radical adducts. However, the trapping efficiency of these nitrone compounds toward lipid alkyl radicals is comparatively low. Consequently, these spin-trapping agents are not applicable to the quantification of lipid alkyl radicals. Five-or six-membered cyclic nitroxyl radicals are relatively stable. Nitroxyl radicals appeared to possess an ability to scavenge carbon-centered radicals (18). This special character of nitroxyl radicals makes it possible to quantitatively trap carbon-centered radicals. Johnson, Caron, and Blough (19) used hydrophilic 3-aminomethyl-2,2,5,5-tetramethylpyrrolidine-N -oxyl to evaluate carbon-centered radical generation: in this method, the adducts were fluorometrically detected after derivatization of amino groups by fluorescamine.The nitroxyl radical-carbon-centered radi...

Toona sinensis is one of the most popular vegetarian cuisines in Taiwan and it has been shown to possess antioxidant, antiangiogenic, and anticancer properties. In this study, we investigated the antiatherosclerotic potential of aqueous leaf extracts from Toona sinensis (TS; 25–100 μg/mL) and its major bioactive compound, gallic acid (GA; 5 μg/mL), in LPS-treated rat aortic smooth muscle (A7r5) cells. We found that pretreatment with noncytotoxic concentrations of TS and GA significantly inhibited inflammatory NO and PGE2 production by downregulating their precursors, iNOS and COX-2, respectively, in LPS-treated A7r5 cells. Furthermore, TS and GA inhibited LPS-induced intracellular ROS and their corresponding mediator, p47phox. Notably, TS and GA pretreatment significantly inhibited LPS-induced migration in transwell assays. Gelatin zymography and western blotting demonstrated that treatment with TS and GA suppressed the activity or expression of MMP-9, MMP-2, and t-PA. Additionally, TS and GA significantly inhibited LPS-induced VEGF, PDGF, and VCAM-1 expression. Further investigation revealed that the inhibition of iNOS/COX-2, MMPs, growth factors, and adhesion molecules was associated with the suppression of NF-κB activation and MAPK (ERK1/2, JNK1/2, and p38) phosphorylation. Thus, Toona sinensis may be useful for the prevention of atherosclerosis.

Lipoxygenases have been proposed to be a possible factor that is responsible for the pathology of certain diseases, including ischaemic injury. In the peroxidation process of linoleic acid by lipoxygenase, the E,Z-linoleate allyl radical-lipoxygenase complex seems to be generated as an intermediate. In the present study, we evaluated whether E,Z-linoleate allyl radicals on the enzyme are scavenged by radical scavengers. Linoleic acid, the content of which was greater than the dissolved oxygen content, was treated with soya bean lipoxygenase-1 (ferric form) in the presence of radical scavenger, CmP (3-carbamoyl-2,2,5,5-tetramethylpyrrolidine-N-oxyl). The reaction rate between oxygen and lipid allyl radical is comparatively faster than that between CmP and lipid allyl radical. Therefore a reaction between linoleate allyl radical and CmP was not observed while the dioxygenation of linoleic acid was ongoing. After the dissolved oxygen was depleted, CmP stoichiometrically trapped linoleate-allyl radicals. Accompanied by this one-electron redox reaction, the resulting ferrous lipoxygenase was re-oxidized to the ferric form by hydroperoxylinoleate. Through the adduct assay via LC (liquid chromatography)-MS/MS (tandem MS), four E,Z-linoleate allyl radical-CmP adducts corresponding to regio- and diastereo-isomers were detected in the linoleate/lipoxygenase system, whereas E,E-linoleate allyl radical-CmP adducts were not detected at all. If E,Z-linoleate allyl radical is liberated from the enzyme, the E/Z-isomer has to reach equilibrium with the thermodynamically favoured E/E-isomer. These data suggested that the E,Z-linoleate allyl radicals were not liberated from the active site of lipoxygenase before being trapped by CmP. Consequently, we concluded that the lipid allyl radicals complexed with lipoxygenase could be scavenged by radical scavengers at lower oxygen content.

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