Fatty acids and vitamins generate singlet oxygen under UVB irradiation.

UVB radiation is already known as initiator and promoter of carcinogenesis in skin. UVB is well absorbed in proteins and DNA leading to products such as cyclobutane pyrimidine dimers. In contrast, UVA radiation generates reactive oxygen species such as singlet oxygen, which can initiate a variety of cellular damages and cellular signalling. It was the goal to investigate whether and to which extent UVB radiation is additionally able to cause oxidative damages via singlet oxygen. Potential endogenous photosensitizers such as vitamin B molecules or unsaturated fatty acids were irradiated in solution using monochromatic UVB radiation at 308 nm. Singlet oxygen was directly detected and quantified by its luminescence at 1270 nm. All investigated endogenous photosensitizers showed clear singlet oxygen signals with a quantum yield ranging from 5 to 40%. UVB radiation altered the photosensitizer molecules during irradiation yielding a change of absorption in the entire ultraviolet spectrum (280-400 nm). UVB irradiation of endogenous photosensitizers produced singlet oxygen that in turn changes the absorption of those molecules. Being an important prerequisite, the changed absorption may either reduce or increase singlet oxygen production. An increase in singlet oxygen generation may initiate a vicious cycle that has the potential to amplify UVB- or UVA-mediated effects in skin cells.

Hydroperoxide characterisation as a signature of the micelle/monomer balance in radiation-induced peroxidation of arachidonate.

Archidonate peroxidation has been studied using HO* radicals radiolytically generated as initiators of this process. Irradiated aqueous solutions of arachidonate (between 0.01 and 25 mM at pH 10.5) have been characterised by means of conjugated dienes measurement (234 nm-absorption spectroscopy) and hydroperoxide detection (high-performance liquid chromatography coupled with a chemiluminescence detection). Radiation-induced peroxidation of arachidonate gives a different trend of peroxide products, depending on the degree of substrate interaction; endoperoxide and hydro-endoperoxide being favored at low concentrations (monomer/oligomer) and monohydroperoxide at high concentrations (micellar form). The experimental ratios G(Hydro2)/G(Hydro1) increase significantly only for arachidonate concentrations higher than 1 mM, i.e. in micellar medium. However, between 0.1 and 1?mM in arachidonate, G-values (for conjugated dienes, Hydro2 and Hydro1) remain nearly constant, meaning that the physical arrangement of the solution changes: Aggregation occurs. The experimental yields of conjugated dienes formation indicated that GDienes > GHO for [arachidonate]>2.5 mM, indicating that a chain propagation process had occurred. Radiolytic yields and structural identification (HPLC-MS analysis) of peroxidation products allowed us to propose a mechanism for the formation of both hydroperoxides.

Arachidonic acid peroxides induce apoptotic Neuro-2A cell death in association with intracellular Ca(2+) rise and mitochondrial damage independently of caspase-3 activation.

The present study aimed at understanding the effects of arachidonic acid peroxides on neuronal cell death using the mouse neuroblastoma cell line, Neuro-2A cells. Arachidonic acid peroxides were produced by ultraviolet (UV) radiation. UV-radiated arachidonic acid significantly reduced Neuro-2A cell viability at concentrations of more than 0.1 muM, with being more potential than non-radiated arachidonic acid. Nuclei of Neuro-2A cells killed with UV-radiated arachidonic acid were reactive to Hoechst 33342, a marker of apoptosis, and the effect was much greater than that achieved with non-radiated arachidonic acid. UV-radiated arachidonic acid persistently increased intracellular Ca(2+) concentrations and dissipated mitochondrial membrane potential in Neuro-2A cells. UV-radiated arachidonic acid-induced Neuro-2A cell death, whereas it was not affected by a pancaspase inhibitor or a caspase-3 inhibitor, was significantly inhibited by an inhibitor of caspase-1, -8, or -9. The results of the present study suggest that arachidonic acid peroxides induce apoptotic neuronal cell death in association with intracellular Ca(2+) rise and mitochondrial damage, in part via a caspase-dependent pathway regardless of caspase-3.

Mechanisms of enhanced apoptosis in HL-60 cells by UV-irradiated n-3 and n-6 polyunsaturated fatty acids.

We examined the effects of arachidonic acid (AA), eicosapentaenoic acid (EPA), and their ultraviolet (UV)-irradiated products on HL-60 cells and isolated mitochondria to explore the following four obscure points in the mechanism of polyunsaturated fatty acids (PUFAs)-induced apoptosis: (i). the role of reactive oxygen species, (ii). the interaction of PUFAs and their metabolites with mitochondria in situ, (iii). the cyclosporine A (CsA)-sensitivity in PUFA-induced membrane permeability transition, (iv). the specificity of oxidized n-3 PUFAs in the induction of apoptosis in cancer cells. UV-oxidized PUFAs contained conjugated dienes and thiobarbituric acid reactive substances (TBARS). The apoptotic effects of PUFAs on HL-60 cells were increased by UV-irradiation whereas the swelling effect of PUFAs on isolated mitochondria was decreased. Both oxidized n-3 and n-6 PUFAs induced increased depolarization, ferricytochrome c release, the activation of various caspases, and DNA-fragmentation in a CsA-insensitive mechanism concomitant with a slight increase in the value of TBARS in cells. Furthermore, there were no significant differences in the mechanism of apoptosis induced by either oxidized AA or oxidized EPA. On the basis of these results, it was concluded that both oxidized n-3 or n-6 PUFAs induced apoptosis in HL-60 cells by a similar mechanism in a CsA-insensitive manner and also that oxidized products of PUFAs, but not the cellular oxidation process itself, play an important role in the mechanism of apoptosis in HL-60 cells.

Cell attachment to extracellular matrices is modulated by pulsed radiation at 820 nm and chemicals that modify the activity of enzymes in the plasma membrane.

BACKGROUND AND OBJECTIVES: Adhesive interactions between cells and extracellular matrices play a regulative role in wound repair processes. The objective of this investigation is to study action mechanisms of pulsed radiation at 820 nm on cellular adhesion in vitro. Light emitting diodes (LED) at 820 nm are widely used for treatment of wounds of various etiology. STUDY DESIGN/MATERIALS AND METHODS: The LED (820 +/- 10 nm, 10 Hz, 16-120 J/m(2)) is used for the irradiation of HeLa cell suspension. In parallel experiments, amiloride (5 x 10(-4) M), ouabain (7 x 10(-5) M, 7 x 10(-4) M), quinacrine (6 x 10(-4) M), arachidonic acid (1 x 10(-5) M), glucose (2 x 10(-4) M), and ATP (5 x 10(-5) M) are added to the cell suspension before or after the irradiation procedure. The cell-glass adhesion is studied using the adhesion assay technique described in Lasers Surg Med 1996; 18:171. RESULTS: Cell-glass adhesion increases in a dose-dependent manner following the irradiation. Preirradiation eliminates the inhibition of cell attachment caused by ouabain, arachidonic acid, and ATP. The inhibitive effect of quinacrine on the cell attachment is eliminated by the irradiation performed after the treatment with the chemical. Irradiation and amiloride have a synergetic stimulative effect on the cell attachment. The threshold dose for the cell attachment stimulation by the irradiation is decreased by the treatment of the cell suspension with amiloride or ouabain. CONCLUSIONS: The results obtained indicate that pulsed IR radiation at 820 nm increases the cell-matrix attachment. It is the modulation of the monovalent ion fluxes through the plasma membrane and not the release of arachidonic acid that is involved in the cellular signaling pathways activated by irradiation at 820 nm. Preirradiation has a protective effect against the inhibitive action of ouabain, arachidonic acid, ATP, and quinacrine on cell attachment process. It is supposed that irradiation activates those signaling pathways in cells which attenuate the inhibitive action of these chemicals.

Stable nitroxide radicals protect lipid acyl chains from radiation damage.

The present study focused on protective activity of two six-membered-ring nitroxide radicals, 2,2,6,6-tetramethylpiperidine-1-oxyl (Tempo) and 4-hydroxy-Tempo (Tempol), against radiation damage to acyl chain residues of egg phosphatidylcholine (EPC) of small unilamellar vesicles (SUV). SUV were gamma-irradiated (10-12 kGy) under air at ambient temperature in the absence and presence of nitroxides. Acyl chain composition of the phospholipids before and after irradiation was determined by gas chromatography. Both Tempo and Tempol effectively and similarly protected the acyl chains of EPC SUV, including the highly sensitive polyunsaturated acyl chains, C20:4, C22:5, and C22:6. The conclusions of the study are: (a) The higher the degree of unsaturation in the acyl chain, the greater is the degradation caused by irradiation. (b) The fully saturated fatty acids palmitic acid (C16) and stearic acid (C18) showed no significant change in their levels. (c) Both Tempo and Tempol provided similar protection to acyl chain residues. (d) Nitroxides' lipid-bilayer/aqueous distribution is not validly represented by their n-octanol/saline partition coefficient. (e) The lipid-bilayer/aqueous partition coefficient of Tempo and Tempol cannot be correlated with their protective effect. (f) The nitroxides appear to protect via a catalytic mode. Unlike common antioxidants, such as alpha-tocopherol, which are consumed under irradiation and are, therefore, less effective against high radiation dose, nitroxide radicals are restored and terminate radical chain reactions in a catalytic manner. Furthermore, nitroxides neither yield secondary radicals upon their reaction with radicals nor act as prooxidants. Not only are nitroxides self-replenished, but also their reduction products are effective antioxidants. Therefore, the use of nitroxides offers a powerful strategy to protect liposomes, membranes, and other lipid-based assemblies from radiation damage.

Kinetics of photoperoxidation of arachidonic acid: molecular mechanisms and effects of antioxidants.

The kinetics of photoperoxidation of [1-14C]arachidonic acid (20:4n-6) at 1.32 mM was studied either with the unsaturated fatty acid alone or in the presence of 10 microM of antioxidants and/or inhibitors of eicosanoid metabolism. The photosensitizer used was meso-tetraphenylporphine. The time-course of the reactions was followed by ultraviolet spectral analysis, thiobarbituric acid reactivity and high-performance liquid chromatographic analysis of aliquots sampled every 15 min during the 4 h of irradiation. The kinetics of photoperoxidation of 20:4n-6 can be divided into three main successive steps: (i) monohydroperoxidation, characterized by the appearance of conjugated diene patterns and monohydroperoxidized 20:4n-6; (ii) secondary oxidation characterized by polyoxygenated products such as dihydroperoxidized 20:4n-6 possessing conjugated triene patterns; and (iii) the disappearance of conjugated patterns and the oxidative cleavage of the products of the two first steps into aldehydic molecules reacting with thiobarbituric acid. During the first 90 min of irradiation, the mechanism of monohydroperoxidation (step one) is purely or predominantly type II photoperoxidation involving only singlet oxygen. This step was inhibited by beta-carotene and by BW755C (3-amino-1-[3-trifluoromethylphenyl]2-pyrazoline). In contrast, the reactions involved in the second and third steps were predominantly type I photoperoxidation involving radical mechanisms. These latter steps were inhibited by beta-carotene, BW755C, vitamin E and probucol. Indomethacin and 5,8,11,14-eicosatetraynoic acid did not alter 20:4n-6 photoperoxidation. This in vitro model of lipid photoperoxidation allows the screening of antioxidants in accordance with their singlet oxygen quenching and/or free radical scavenging properties.