Inhibition of immediate type hypersensitivity reaction by combined irradiation with ultraviolet and visible light

Recently we found that ultraviolet B (UVB) irradiation in erythematous doses significantly inhibited the immediate type hypersensibility reaction in the skin. In the present study we investigated the effects of different wavelengths on the skin prick test reaction (SPT). The forearm of ragweed allergic patients was irradiated with increasing doses of ultraviolet A (UVA), visible light (VIS) or combined UVB, UVA and VIS light, referred to as mUV/VIS. SPTs were performed 24 h after irradiation both on irradiated and non-irradiated control skin areas using ragweed extract. UVA and VIS irradiation led to a slight, not significant inhibition of allergen-induced wheal formation. Mixed irradiation with mUV/VIS light resulted in a dose-dependent inhibition of the allergen-induced wheal formation. The inhibition was significant already at suberythematous doses. As there is a good correlation between SPT and the nasal symptoms in patients with hay fever these data suggest that phototherapy with mUV/VIS light might be an effective and safe treatment modality for immediate type hypersensibility reactions in the skin and nasal mucosa.     

Influence of UVB,UVA and UVA1 irradiation on histamine release from human basophils and mast cells in vitro in the presence and absence of antioxidants

UV irradiation is widely used for the treatment of atopic eczema. In recent years, UVA1 phototherapy has gained increasing attention. This study analyzed the influence of different UV wavelengths--especially UVA1--on histamine release from human basophils and mast cells. The modulation of this parameter might be responsible for some of the therapeutic effects of UV irradiation. Enriched human basophils and human mast cells (HMC1 cell line) were irradiated with increasing doses of UVB, UVA and UVA1 in vitro. After irradiation, different stimulants were added to induce histamine release. In additional experiments, basophils were preincubated with superoxide dismutase, ascorbate or trolox to study the role of antioxidants in the modulation of histamine release after UV irradiation. UVA and UVA1 significantly inhibited histamine release from basophils and mast cells. UVB only had an inhibitory effect on mast cells. Preincubation with superoxide dismutase and ascorbate did not influence the inhibitory effect of UVA1 on basophil histamine release, whereas trolox decreased significantly the histamine release from nonirradiated basophils.     

Effect of UVA radiation on membrane fluidity and radical decay in human fibroblasts as detected by spin labeled stearic acids

The ultraviolet A (UVA) radiation component of sunlight (320-400 nm) has been shown to be a source of oxidative stress to cells via generation of reactive oxygen species. We report here some consequences of the UVA irradiation on cell membranes detected by electron paramagnetic resonance (EPR) spectroscopy. Paramagnetic nitroxide derivatives of stearic acid bearing the monitoring group at different depths in the hydrocarbon chain were incorporated into human fibroblasts membranes to analyze two main characteristics: kinetics of the nitroxide reduction and membrane fluidity. These two characteristics were compared for control and UVA-irradiated (0-250 kJ/m(2)) cells. The term relative redox capacity (RRC) was introduced to characterize and to compare free radical reduction measured by EPR with some well-known viability/clonogenicity tests. Our results showed that UVA-irradiation produces a more rigid membrane structure, especially at higher doses. Furthermore, we found that trends agree in survival measured by neutral red (NR), trypan blue (TB), and clonogenic efficiency compared with RRC values measured by EPR for low and medium exposure doses. Above 100 kJ/m(2), differences between these tests were observed. Antioxidant effect was modeled by alpha-tocopherol-acetate treatment of the cells before UVA irradiation. While NR, TB and clonogenicity tests showed protection at the highest UVA doses (>100 kJ/m(2)), results obtained with EPR measurements, both membrane fluidity and kinetics, or using MTT test did not exhibit this protective effect.     

In vitro phototoxicity of nifedipine: sequential induction of toxic and non-toxic photoproducts with UVA radiation.

Anecdotal reports suggest that the dihydropyridine calcium antagonist, nifedipine (NIF), may be phototoxic in human skin. We have studied NIF phototoxicity in vitro using UVA fluorescent tubes (Sylvania PUVA). NIF was phototoxic to Candida albicans and induced photohaemolysis both with NIF present during irradiation and with pre-irradiated drug. In V79 hamster fibroblasts, NIF (10 micrograms ml-1) was phototoxic MTT assay) 24 h after irradiation (0-112 kJ m-2); at 7.5 kJ m-2, about 70% of cells were damaged whilst at 37.5 kJ m-2, only about 45% of cells were damaged. A similar pattern was seen with pre-irradiated NIF. Absorption spectroscopy showed that the NIF absorption maximum (Amax approximately 340 nm) blue-shifted to 314 nm at low UVA doses (7.5 kJ m-2 or less) and red-shifted to 345 nm at higher doses (isosbestic point, 325 nm). Thin layer chromatography of irradiated NIF showed a single photoproduct (PP1; Amax approximately 314 nm) formed at 7.5 kJ m-2 or less which disappeared at higher UVA doses to give further photoproducts. PP1 was highly dark toxic to V79 cells (50% damage at about 5 micrograms ml-1) but PP1 pre-irradiated with UVA was non-toxic. Preliminary gas chromatography-mass spectroscopy studies suggest that PP1 is the nitroso derivative of NIF. These results indicate that NIF phototoxicity in vitro is partially mediated by initial formation of a toxic photoproduct (PP1) but, paradoxically, subsequent UVA irradiation may reduce phototoxicity. The NIF concentrations required to induce in vitro phototoxicity are much greater than therapeutic plasma levels. Unless there is skin accumulation of NIF or PP1, our in vitro results suggest that NIF may not be an important skin-photosensitizing agent in vivo.     

Catalyst‐Free Photoredox Addition–Cyclisations: Exploitation of Natural Synergy between Aryl Acetic Acids and Maleimide

Suitably functionalised carboxylic acids undergo a previously unknown photoredox reaction when irradiated with UVA in the presence of maleimide. Maleimide was found to synergistically act as a radical generating photoxidant and as a radical acceptor, negating the need for an extrinsic photoredox catalyst. Modest to excellent yields of the product chromenopyrroledione, thiochromenopyrroledione and pyrroloquinolinedione derivatives were obtained in thirteen preparative photolyses. In situ NMR spectroscopy was used to study each reaction. Reactant decay and product build‐up were monitored, enabling reaction profiles to be plotted. A plausible mechanism, whereby photo‐excited maleimide acts as an oxidant to generate a radical ion pair, has been postulated and is supported by UV/Vis. spectroscopy and DFT computations. The radical‐cation reactive intermediates were also characterised in solution by EPR spectroscopy.     

Photochemical and phototoxic properties of ethyl 1,4-dihydro-8-nitro-4-oxoquinoline-3-carboxylate, a new quinoline derivative

The present study demonstrates photoinduced generation of superoxide radical anion and singlet oxygen upon UVA irradiation of ethyl 1,4-dihydro-8-nitro-4-oxoquinoline-3-carboxylate (DNQC), and its cytotoxic/phototoxic effects on murine leukemia L1210 cells. The formation of reactive oxygen species (ROS) was investigated by EPR spectroscopy using in situ spin trapping technique and 4-hydroxy-2,2,6,6-piperidine (TMP) for singlet oxygen ((1)O(2)) detection. The EPR spectra monitored upon photoexcitation of aerated solutions of DNQC in dimethylsulfoxide evidenced the efficient activation of molecular oxygen via Types I and II mechanisms. The cytotoxic/phototoxic effects of DNQC, analysis of cell cycle, induction of apoptosis/necrosis, DNA damage and molecular mechanism of apoptotic death of L1210 cells in dark and in the presence of UVA irradiation were compared. DNQC induced a different cytotoxic/phototoxic effect, which was concentration- and time-dependent. The four highest tested concentrations of non-photoactivated and photoactivated DNQC induced immediate cytotoxic/phototoxic effect after 24h cultivation of L1210 cells. This effect decreased with the time of treatment. The irradiation increased the sensitivity of leukemia cell line on DNQC, but the cell sensitivity decreased with time of processing. Quinolone derivative DNQC significantly induced direct DNA strand breaks in L1210 cells, which were increased with the irradiation of cells. The DNA damage generated by DNQC alone/with combination of UVA irradiation induced cell arrest in G(0)/G(1) and G(2)/M phases, decrease in the number of L1210 cells in Sphase and apoptotic cell death of certain part of cell population after 24 h of influence. DNQC alone/with combination of UVA irradiation induced apoptosis in L1210 cells through ROS-dependent mitochondrial pathway.     

LET effects following neutron irradiation of lithium formate EPR dosimeters

Lithium formate electron paramagnetic resonance (EPR) dosimeters were irradiated using 60Co gamma-rays or fast neutrons to doses ranging from 5 to 20 Gy and investigated by EPR spectroscopy. Using a polynomial fitting procedure in order to accurately analyze peak-to-peak line widths of first derivative EPR spectra, dosimeters irradiated with neutrons had on average 4.4+/-0.9% broader EPR resonance lines than gamma-irradiated dosimeters. The increase in line width was slightly asymmetrical. Computer simulated first derivative polycrystalline EPR spectra of a *CO2- radical gave very good reconstructions of experimental spectra of irradiated dosimeters. The spectrum simulations could then be used as a tool to investigate the line broadening observed following neutron irradiation. It was shown that an increase in the simulated Lorentzian line width could explain both the observed line broadening and the asymmetrical effect. The ratio of the peak-to-peak amplitude of first derivative EPR spectra obtained at two different microwave powers (20 and 0.5 mW) was 7.8+/-1.2% higher for dosimeters irradiated with neutrons. The dependence of the spectrum amplitude on the microwave power was extensively investigated by fitting observations to an analytical non-linear model incorporating, among others, the spin-lattice (T1) and spin-spin (T2) relaxation times as fitting parameters. Neutron irradiation resulted in a reduction in T(2) in comparison with gamma-irradiation, while a smaller difference in T1 was found. The effects observed indicate increased local radical density following irradiation using high linear energy transfer (LET) neutrons as compared to low LET gamma-irradiation. A fingerprint of the LET may thus be found either by an analysis of the line width or of the dependence of the spectrum amplitude on the microwave power. Lithium formate is therefore a promising material for EPR dosimetry of high LET radiation.     

Comparative identification of irradiated herbs by the methods of electron paramagnetic resonance and thermoluminescence

Non irradiated and γ-irradiated dry herbs savoury (Savoury), wild thyme (Thymus serpollorium) and marjoram (Origanum) with absorbed dose of 8 kGy have been investigated by the methods of elecrtron paramagnetic resonance (EPR) and thermoluminescence (TL). Non-irradiated herbs exhibit only one weak siglet EPR signal whereas in irradiated samples its intensity increase and in addition two satelite lines are recorded. This triplet EPR spectrum is attributed to cellulose free radical generated by irradiation. It has been found that upon keeping the samples under the normal stock conditions the life-time of the cellulose free radical in the examined samples is ∼60–80 days. Thus the conclusion has been made that the presence of the EPR signal of cellulose free radical is unambiguous indication that the sample under study has been irradiated but its absence can not be considered as the opposite evidence. In the case when EPR signal was absent the method of TL has been used to give the final decision about the previous radiation treatment of the sample.     

Photochemical and phototoxic activity of berberine on murine fibroblast NIH-3T3 and Ehrlich ascites carcinoma cells

The present study demonstrates photoinduced generation of superoxide anion radical and singlet oxygen upon UVA irradiation of berberine chloride, and its cytotoxic/phototoxic effects on murine fibroblast non-cancer NIH-3T3 and Ehrlich ascites carcinoma (EAC) cells. The EPR spectra monitored upon photoexcitation of aerated solutions of berberine evidenced the efficient activation of molecular oxygen via Type I and II mechanisms, as the generation of superoxide anion radical and singlet oxygen was observed. The EAC cell line was more sensitive to the effect of non-photoactivated and photoactivated berberine than the NIH-3T3 cell line. UVA irradiation increased the sensitivity of EAC cells to berberine, while the sensitivity of NIH-3T3 cells to photoactivated berberine was not changed. Berberine significantly induced direct DNA strand breaks in tested cells, oxidative lesions were not detected, and the effect of irradiation of cells after berberine treatment did not affect the increase of DNA damage in EAC and NIH-3T3 cells. The DNA damage generated by a combination of berberine with UVA irradiation induced a significant blockage of EAC cells in the S and G(2)/M phases and the stopping/decrease of cell proliferation after 24h of influence. On the other hand, after 36h or 48h of berberine treatment, the DNA damage induced necrotic or apoptotic death of EAC cells. Whether these divergences are caused by differences in the properties of two non-isogenic cell lines or by different berberine uptake and cell localization will be analyzed in our further investigations.     

High performance liquid chromatography/electron spin resonance/mass spectrometry analyses of radicals formed in an anaerobic reaction of 9- (or 13-) hydroperoxide octadecadienoic acids with ferrous ions

The oxidation of linoleic acid yields isomeric acyl hydroperoxides. In order to clarify the relation between the lipid peroxide-derived radicals and the toxicity of the lipid peroxide, identification of the lipid-derived radicals is essential. In this paper, high performance liquid chromatography/electron spin resonance/mass spectrometry (HPLC/EPR/MS) analysis of the radicals was performed for the reaction mixture containing 9-hydroperoxy-(10E,12E)-octadeca-10,12-dienoic acid (9EE-OOH) [or 13-hydroperoxy-(9Z,11E)-octadeca-9,11-dienoic acid (13ZE-OOH)] under an aerobic condition or an anaerobic condition. Following radicals were identified from 9EE-OOH (or 13EZ-OOH) by using high performance liquid chromatography/electron spin resonance spectrometry (HPLC/EPR) and HPLC/EPR/MS: pentyl radical and isomers of epoxylinoleic acid radicals from 13EZ-OOH under an anaerobic condition; 7-carboxyheptyl radical and pentyl radical from 13EZ-OOH under an aerobic condition; 7-carboxyheptyl radical and pentyl radical from 9EE-OOH under an aerobic condition; 7-carboxyheptyl radical from 9EE-OOH under an anaerobic condition. These results showed that the formation of the respective radical species depends on oxygen concentration in the reaction mixtures to a great extent.     

Quantitation of the Reactive Oxygen Species Generated by the UVA Irradiation of Ascorbic Acid-Glycated Lens Proteins

The oxidation products of ascorbic acid rapidly glycate proteins and produce protein-bound, advanced glycation endproducts. These endproducts can absorb UVA light and cause the photolytic oxidation of proteins (Ortwerth, Linetsky and Olesen, Photochem. Photobiol . 62, 454–463, 1995), which is mediated by the formation of reactive oxygen species. A dialyzed preparation of calf lens proteins, which had been incubated for 4 weeks with 20 mM ascorbic acid in air, was irradiated for 1 h with 200 mW/ cm² of absorbed UVA light (λ > 338 nm), and the concentration of individual oxygen free radicals was measured. Superoxide anion attained a level of 76 μ M as determined by the superoxide dismutase (SOD)-depen-dent increase in hydrogen peroxide formation and of 52 μ M by the SOD-inhibitable reduction of cytochrome c. Hydrogen peroxide formation increased linearly to 81 μM after 1 h. Neither superoxide anion nor hydrogen peroxide, however, could account for the UVA photolysis of Trp and His seen in this system.
Singlet oxygen levels approached 1.0 mM as measured by the oxidation of histidine, which was consistent with singlet oxygen measurements by the bleaching of N,N- dimethyl-4-nitrosoaniline. High concentrations of sodium azide, a known singlet oxygen quencher, inhibited the photolytic destruction of both His and Trp. Little or no protein damage could be ascribed to hydroxyl radical based upon quenching experiments with added mannitol. Therefore, superoxide anion and H₂O₂ were generated by the UVA irradiation of ascorbate advanced glycation endproducts, however, the major reactive oxygen species formed was singlet oxygen.     

Microstructural studies of electron beam-irradiated cellulose pulp

The effect of electron beam irradiation on the microstructure of cellulose has been investigated using positron annihilation lifetime spectroscopy (PALS) and electron paramagnetic resonance (EPR) Spectroscopy. PALS studies of irradiated cellulose samples showed that ortho-positronium (o-Ps) lifetime increases with an increase in dose up to 80 kGy and decreases at higher doses. The EPR signal of the irradiated cellulose matrix showed the presence of multiple radical sites. These results are discussed on the basis of chemical and physical changes occurring at the microscopic level in the cellulose due to irradiation.     

Sucrose radical-production cross-section regarding heavy-ion irradiation

We investigated the sucrose radical-production cross-section induced by heavy-ion irradiation. L-alanine was also used in order to compare radical yield and cross-section. The stable free radicals after irradiation were analyzed by electron paramagnetic resonance (EPR). The radical yield obtained by the irradiated samples had a logarithmic correlation with the LET (linear energy transfer). Quantitative EPR analyses showed that radical productions for sucrose and L-alanine vary both by different particle irradiation and the LET under the same absorbed dose. Furthermore, the cross-sections of radical productions for samples were calculated. Both cross-sections for C ions irradiation under LET 30 keV/microm at 50 Gy dose were approximately 3.0x10(-9) microm(2), taking account of the molecular areas of the samples. The values of the cross-sections imply that multiple ionizing particles involve producing stable radicals.     

Structures and photoactive properties of poly(styrene‐co‐vinylbenzophenone)

Poly(styrene‐co‐vinylbenzophenone) prepared by a graft reaction on polystyrene revealed photoactive properties under irradiation of UVA. The photoactive structural features of the polymer were examined via electron paramagnetic resonance (EPR) under irradiation of UVA and fluorescent light. The photoactive functions of the polymer such as antimicrobial performance and dye decolorization ability were investigated. The results revealed that the poly(styrene‐co‐vinylbenzophenone) could generate radicals under fluorescent and UVA irradiation, and some radicals could stay alive for about 30 min in a dark chamber. The photoexcited polymer showed excellent antibacterial ability and decolorization effect on methylene blue and methyl orange dye under both daylight and UVA light. © 2008 Wiley Periodicals, Inc. J Polym Sci Part B: Polym Phys 46: 2423–2430, 2008     

Effect of Two Different UVA Doses on the Rabbit Cornea and Lens

The aim of the present paper was to examine the irradiation effect of two doses of UVA rays (365 nm) on the rabbit cornea and lens. Corneas of anesthetized adult albino rabbits were irradiated with UVA rays for 5 days (daily dose 1.01 J cm⁻² in one group of rabbits and daily dose 2.02 J cm⁻² in the second group of animals). The third day after the last irradiation, the rabbits were killed, and their eyes were employed for spectrophotometrical, biochemical and immunohistochemical investigations. Normal eyes served as controls. Absorption spectra of the whole corneal centers were recorded over the UV–VIS (visible) spectral range. Levels of antioxidant and prooxidant enzymes, nitric oxide synthases and nitric oxide (indirectly measured as nitrate concentration) were investigated in the cornea. Malondialdehyde, a byproduct of lipid peroxidation, was examined in the cornea and lens. The results show that the staining for endothelial nitric oxide synthase was more pronounced in corneas irradiated with the higher UVA dose. Otherwise, UVA rays at either dose did not significantly change corneal light absorption properties and did not cause statistically significant metabolic changes in the cornea or lens. In conclusion, UVA rays at the employed doses did not evoke harmful effects in the cornea or lens.     

Red Light Interferes in UVA‐Induced Photoaging of Human Skin Fibroblast Cells

The possible regulation mechanism of red light was determined to discover how to retard UVA‐induced skin photoaging. Human skin fibroblasts were cultured and irradiated with different doses of UVA, thus creating a photoaging model. Fibroblasts were also exposed to a subtoxic dose of UVA combined with a red light‐emitting diode (LED) for five continuous days. Three groups were examined: control, UVA and UVA plus red light. Cumulative exposure doses of UVA were 25 J cm^?2, and the total doses of red light were 0.18 J cm^?2. Various indicators were measured before and after irradiation, including cell morphology, viability, β‐galactosidase staining, apoptosis, cycle phase, the length of telomeres and the protein levels of photoaging‐related genes. Red light irradiation retarded the cumulative low‐dose UVA irradiation‐induced skin photoaging, decreased the expression of senescence‐associated β‐galactosidase, upregulated SIRT1 expression, decreased matrix metalloproteinase MMP‐1 and the acetylation of p53 expression, reduced the horizon of cell apoptosis and enhanced cell viability. Furthermore, the telomeres in UVA‐treated cells were shortened compared to those of cells in the red light groups. These results suggest that red light plays a key role in the antiphotoaging of human skin fibroblasts by acting on different signaling transduction pathways.     

Singlet Oxygen–mediated Hydroxyl Radical Production in the Presence of Phenols: Whether DMPO–·OH Formation Really Indicates Production of ·OH?¶

The reaction of singlet oxygen (1O2) generated by ultraviolet-A (UVA)-visible light (lambda > 330 nm) irradiation of air-saturated solutions of hematoporphyrin with phenolic compounds in the presence of a spin trap, 5,5-dimethyl-1-pyrroline-N-oxide (DMPO), gave an electron spin resonance (ESR) spectrum characteristic of the DMPO-hydroxyl radical spin adduct (DMPO-*OH). In contrast, the ESR signal of 5,5-dimethyl-2-pyrrolidone-N-oxyl, an oxidative product of DMPO, was observed in the absence of phenolic compounds. The ESR signal of DMPO-*OH decreased in the presence of either a *OH scavenger or a quencher of *O2 and under anaerobic conditions, whereas it increased depending on the concentration of DMPO. These results indicate both 1O2- and DMPO-mediated formation of free *OH during the reaction. When DMPO was replaced with 5-(diethoxyphosphoryl)-5-methyl-1-pyrroline-N-oxide (DEPMPO), no DEPMPO adduct of oxygen radical species was obtained. This suggests that 1O2, as an oxidizing agent, reacts little with DEPMPO, in which a strong electron-withdrawing phosphoryl group increases the oxidation potential of DEPMPO compared with DMPO. A linear correlation between the amounts of DMPO-*OH generated and the oxidation potentials of phenolic compounds was observed, suggesting that the electron-donating properties of phenolic compounds contribute to the appearance of *OH. These observations indicate that 1O2 reacts first with DMPO, and the resulting DMPO-1O2 intermediate is immediately decomposed/reduced to give *OH. Phenolic compounds would participate in this reaction as electron donors but would not contribute to the direct conversion of 1O2 to *OH. Furthermore, DEPMPO did not cause the spin-trapping agent-mediated generation of *OH like DMPO did.     

Electron beam radiation effects on UHMWPE: an EPR study

Electron paramagnetic resonance (EPR) technique has been employed to detect and characterise a series of different radical species generated in ultra‐high molecular weight polyethylene (UHMWPE) via electron beam irradiation. Three different radical species have been found and assigned on the basis of their EPR spectra and of the related computer simulations. A secondary alkyl species, the prevalent one, is present immediately after irradiation, an allyl species appears only 24 h after irradiation when the alkyl species disappears.The third species, clearly visible at high microwave power only, has been observed for the first time and assigned to a tertiary alkyl carbon radical, whose formation is strictly connected with a Y‐shape crosslink and a migration of the unpaired electron on a carbon atom localised in an adjacent position. Copyright © 2011 John Wiley & Sons, Ltd.     

Lifetime of alkyl macroradicals in irradiated ultra-high molecular weight polyethylene

The interaction of UHMWPE with an electron beam in vacuum and in the presence of oxygen has been investigated. UHMWPE irradiated to various doses was examined with Electron Paramagnetic Resonance (EPR) and Fourier Transform infra-red (FTIR) spectroscopies. EPR was used to explore the nature of radicals produced in UHMWPE upon irradiation and to follow their decay as a function of time. Hydroperoxides formation and distribution throughout the samples were studied with FTIR spectroscopy.A correlation between the rate of decay of macroradicals and that of hydroperoxide formation has been proposed. Accordingly, the lifetime of secondary alkyl macroradicals in the amorphous phase of the polymer was found to range approximately from 3 to 10 h.     

The photochemistry of lipoic acid: photoionization and observation of a triplet excited state of a disulfide.

Under short-wavelength UV irradiation, lipoic acid (LipSS) and its reduced form, dihydrolipoic acid (DHLA), undergo photoionization processes through a bi- or monophotonic pathway. After ionization, the LipSS radical cation (LipSS*+) and radical anion (LipSS*-) are generated. LipSS*- can be converted to equimolar amounts of LipSS and DHLA through second-order decay. Triplet acetone can be quenched by LipSS and DHLA with a rate close to the diffusion-controlled limit. The mechanism was further confirmed by continuous irradiation experiments. When LipSS is directly irradiated with UVA light, the first excited triplet state of LipSS is observed, with a lifetime tau=75 ns. Characteristic reactions include triplet energy transfer to oxygen and beta-carotene and addition to isoprene. The lifetime of triplet LipSS is also shortened by addition of water and methanol.