ULTRAVIOLET LIGHT-INDUCED FREE RADICAL FORMATION IN SKIN: AN ELECTRON PARAMAGNETIC RESONANCE STUDY

It has been suggested that ultraviolet light induces free radical formation in skin, leading to photoaging and cancer. We have demonstrated by electron paramagnetic resonance that the ascorbate free radical is naturally present in unexposed skin at a very low steady state level. When a section of SKH-1 hairless mouse skin in an EPR cavity is exposed to UV light (4,500 J m⁻²−1, Xe lamp, 305 nm cutoff and IR filters), the ascorbate free radical signal intensity increases. These results indicate that UV light increases free radical oxidative stress, consistent with ascorbate's role as the terminal, small-molecule antioxidant. The initial radicals produced by UV light would have very short lifetimes at room temperature; thus, we have applied EPR spin trapping techniques to detect these radicals. Using α-[4-pyridyl 1-oxide]-N- tert -butyl nitrone (POBN), we have for the first time spin trapped a UV light-produced carbon-centered free radical from intact skin. The EPR spectra exhibited hyperfine splittings that are characteristic of POBN/alkyl radicals, a^N= 15.56 G and a^H= 2.70 G, possibly generated from membrane lipids as a result of β-scission of lipid alkoxyl radicals. Iron can act as a catalyst for free radical oxidative reactions; chronic exposure of skin to UV radiation causes increased iron deposition. Using our spin trapping system, we have shown that topical application of the iron-chelator, Desferal, to a section of skin reduces the UV light-induced POBN adduct radical signal. These results provide direct evidence for free radical generation and a role for iron in UV light-induced dermatopathology. We suggest that iron chelators can serve as photoprotective agents by preventing these oxidations.     

Role of radicals in UV‐initiated postplasma grafting of poly‐ε‐caprolactone: An electron paramagnetic resonance study

Electron paramagnetic resonance (EPR) measurements were performed on poly‐ε‐caprolactone (PCL) films at different stages of the postplasma‐grafting process. PCL films prepared by solvent casting (SC) or electrospinning (ESP) yield very similar EPR spectra after Ar‐plasma treatment and subsequent exposure to air, but the EPR signal is much stronger in the PCL‐ESP films. The free radicals appear to be mainly, and possibly exclusively, oxygen centered. The radicals generated by UV irradiation in PCL‐ESP films were studied in situ with EPR, using a UV‐LED (λ = (285 ± 5) nm). Their EPR spectrum is distinctly different from the plasma‐induced signal, indicative of carbon‐centered radicals, and appears to be independent of the plasma pretreatment. UV‐induced homolytic splitting of (hydro)peroxide bonds was not observed. Both the plasma‐ and UV‐induced radicals decay at room temperature (RT), even in an inert atmosphere. This study demonstrates the potential of electrospun films and UV‐LEDs for the study of plasma‐ and UV‐generated free radicals with EPR in polyesters, and raises questions with respect to the validity of some generally accepted molecular mechanisms underpinning the postplasma grafting technique for polyesters. © 2012 Wiley Periodicals, Inc. J Polym Sci Part A: Polym Chem, 2012     

EPR Detection of Free Radicals in UV-lrradiated Skin: Mouse Versus Human

Abstract— Ultraviolet radiation produces free radicals in Skh-1 mouse skin, contributing to photoaging and carcinogenesis. If a mouse model is a general indicator of free radical processes in human skin photobiology, then radical production observed in mouse and human skin should be directly comparative. In this work we show that UV radiation (Λ > 300 nm, 14 μW/cm² UVB; 3.5 mW/cm² UVA) increases the ascorbate free radical (Asc^.-) electron paramagnetic resonance (EPR) signal in both Skh-1 mouse skin (45%) and human facial skin biopsies (340%). Visible light (Λ > 400 nm; 0.23 mW/cm² UVA) also increased the Asc^.- signal in human skin samples (45%) but did not increase baseline mouse Asc^.-, indicating that human skin is more susceptible to free radical formation and that a chromophore for visible light may be present. Using EPR spin-trapping techniques, UV radiation produced spin adducts consistent with trapping lipid alkyl radicals in mouse skin (α-[4-pyridyl 1-oxide]-N-tert-butyl nitrone/alkyl radical adduct; a^N= 15.56 G and a^H= 2.70 G) and lipid alkoxyl radicals in human skin (5,5-dimethylpyrroline-l-oxide/alkoxyl radical adduct; a^N= 14.54 G and a^H= 16.0 G). Topical application of the iron chelator Desferal to human skin significantly decreases these radicals (±50%), indicating a role for iron in lipid peroxidation; Desferal has previously been shown to decrease radical production in mouse skin. This work supports the use of the Skh-1 mouse as a predictive tool for free radical formation in human skin. These results provide the first direct evidence for UV radiation-induced free radical formation at near physiological temperatures in human skin and suggest that iron chelators may be useful as photoprotective agents.     

EPR study of light illumination effects on radicals in gamma-irradiated L-alanine

Exposure of gamma-irradiated L-alanine samples to sunlight and to light from a regular, fluorescent lamp resulted in significant changes in their EPR resonance patterns, both to spectral shapes and intensities. The experimental EPR spectra were numerically decomposed into three components reflecting contributions of three different radicals (R1-R3) generated by ionizing radiation in alanine. The light exposure caused a decay of the measured EPR signal intensity. For similar light intensities and exposure times the decay was much more pronounced in samples illuminated by sunlight than in samples illuminated by the fluorescent lamp. In both cases light-induced decay of R1 radicals was observed. Sunlight illumination resulted in a moderate decay of R2 radicals and in a doubling of the R3 radical population. On the other hand, fluorescent light caused a significant increase of R2 radicals and did not change the amount of R3 radicals. A quantitative analysis of the variations of the three radical contributions to the total EPR spectra upon fluorescent light exposure suggests a net R1-->R2 free radical transformation. These effects of light on the alanine dosimetric signal should be taken into account in dosimetry protocols, assuring protection of alanine dosimeters from extended exposure to light.     

The role of melanin as protector against free radicals in skin and its role as free radical indicator in hair

Throughout the body, melanin is a homogenous biological polymer containing a population of intrinsic, semiquinone-like radicals. Additional extrinsic free radicals are reversibly photo-generated by UV and visible light. Melanin photochemistry, particularly the formation and decay of extrinsic radicals, has been the subject of numerous electron spin resonance (ESR) spectroscopy studies. Several melanin monomers exist, and the predominant monomer in a melanin polymer depends on its location within an organism. In skin and hair, melanin differs in content of eumelanin or pheomelanin. Its bioradical character and its susceptibility to UV irradiation makes melanin an excellent indicator for UV-related processes in both skin and hair. The existence of melanin in skin is strongly correlated with the prevention against free radicals/ROS generated by UV radiation. Especially in the skin melanin (mainly eumelanin) ensures the only natural UV protection by eliminating the generated free radicals/ROS. Melanin in hair can be used as a free radical detector for evaluating the efficacy of hair care products. The aim of this study was to investigate the suitability of melanin as protector of skin against UV generated free radicals and as free radical indicator in hair.     

An in vivo study of free radicals generated in murine skin by protoporphyrin IX and visible light

Lipids extracted from the skin of C57BL/6J mice injected subcutaneously with alpha-(4-pyridyl-1-oxide)-N-tert-butylnitrone (POBN) and exposed to topical protoporphyrin IX (PPIX) and visible light had significantly higher levels of POBN spin adducts compared with dark PPIX exposed or vehicle-treated controls. Computer analysis of the POBN adduct electron paramagnetic (spin) resonance (EPR) spectra indicated that two radical species were present in each extract, one of which was a lipid-derived carbon-centered adduct (1, a(N) = 14.8 G and a(H) = 2.6 G), whereas the other (2, a(N) = 13.8 G and a(H) = 1.8 G) was probably oxygen centered. Adduct 2 was present in greater proportion in lipids extracted from PPIX/light-exposed mice compared with dark or vehicle-treated controls. These findings suggest that PPIX/light generates free radicals in mouse skin, thus providing a radical mechanism for PPIX-induced photosensitivity. Our approach may be useful for the detection of free radicals generated by other skin photosensitizers and may also provide a means for testing putative skin-protecting agents.     

Measurements of UV-generated free radicals/reactive oxygen species (ROS) in skin

Free radicals/reactive oxygen species (ROS) generated in skin by UV irradiation were measured by electron spin resonance (ESR). To increase the sensitivity of measurement the short life free radicals/ROS were scavenged and accumulated by using the nitroxyl probe 3-carboxy-2,2,5,5-tetrametylpyrrolidine-1-oxyl (PCA). The spatial distribution of free radicals/ROS measured in pig skin biopsies with ESR imaging after UV irradiation corresponds to the intensity decay of irradiance in the depth of the skin. The main part of free radicals/ROS were generated by UVA (320-400 nm) so that the spatial distribution of free radicals reaches up to the lower side of the dermis. In vivo measurements on human skin were performed with a L-band ESR spectrometer and a surface coil integrating the signal intensities from all skin layers to get a sufficient signal amplitude. Using this experimental arrangement the protection of UVB and UVA/B filter against the generation of free radicals/ROS in skin were measured. The protection against ROS and the repair of damages caused by them can be realized with active antioxidants characterized by a high antioxidative power (AP). The effect of UV filter and antioxidants corresponding to their protection against free radicals/ROS in skin generated by UVAB irradiation can be quantified by the new radical sun protection factor (RSF). The RSF indicates the increase of time for staying in the sun to generate the same number of free radicals/ROS in the skin like for the unprotected skin. Regarding the amount of generated free radicals/ROS in skin as an biophysical endpoint the RSF characterizes both the protection against UVB and UVA radiation.     

Study of compounds suppressing free radical generation from UV-exposed ketoprofen

Ketoprofen [(RS)-2-(3-benzoylphenyl)propanoic acid] is widely used for the treatment of inflammatory diseases and musculoskeletal injury. However, there is concern regarding its potential for photosensitization as a side effect. Free radicals and active oxygen species generated from ketoprofen on exposure to ultraviolet (UV) light have been implicated in phototoxicity and photosensitization. In this study, we examined the suppressing ability of some compounds for the free radicals and active oxygen species generated by the photodynamic reaction of ketoprofen, to determine a new resist of photosensitization by ketoprofen. Eight compounds, including six known free radical scavengers were individually mixed with ketoprofen, and the mixtures were exposed to UV. Then, the free radicals and the active oxygen species were determined by the electron spin resonance spectrometry to estimate suppressing and scavenging ability of compounds. The compounds that show promise in suppressing superoxide anion generation from UV-exposed ketoprofen were further evaluated using the on-line photo-irradiated superoxide anion detection system. It was confirmed that quercetin, a flavonoid, strongly suppresses the generation of free radicals and active oxygen species from UV-exposed ketoprofen. The experiments using the experimental formulation of an adhesive skin patch of ketoprofen containing quercetine and the Chemiluminescence analyzer (CLA) indicated that quercetin has high potential for use as an excipient in ketoprofen ointments to suppress phototoxicity and photosensitization by ketoprofen.     

The effect of dose on light-sensitivity of radicals in alanine EPR dosimeters

In this work we present a study of light-induced effects on free radicals and their transformations in gamma-irradiated pure L-alanine and in commercially available alanine detectors: rods, pellets and films. Samples irradiated to doses from 2 Gy to 4000 kGy were exposed to light from a fluorescent lamp and to ordinary daylight. The observed changes in EPR spectra of the samples were analyzed with regard to their intensity and shape. The shape analysis was based on numerical decomposition of the measured spectra into model spectra reflecting contributions of R1, R2 and R3 radical populations in the samples. The illumination of alanine dosimeters resulted in significant decrease of the central EPR line and was accompanied by distinct variations in the shape of EPR spectra. The rate of light-induced decay in spectra amplitude was found to be dependent on dose of ionizing radiation--the sensitivity to light was decreasing with increase in dose in all detectors in the 2-5x10(5) Gy dose range. The exposure of gamma-irradiated (to 300 Gy) alanine to normal, diffused daylight resulted in decay of the signal amplitude at rate about 0.5% per week. It was shown that decay in the R1 component was responsible for the observed reduction of the spectra amplitude. The observed increase in R2 contributions in samples exposed to light confirmed a hypothesis of R-->R2 radical transformations promoted by visible light. The reported effects indicate a necessity of protection of irradiated dosimeters from their prolonged exposure to light.     

PHOTOCHEMISTRY OF 2-MERCAPTOPYRIDINES. PART 3. EPR STUDY OF PHOTOPRODUCTION OF HYDROXYL RADICALS BY N-HYDROXYPYRIDINE-2-THIONE USING 5,5-DIMETHYL-1-PYRROLINE N-OXIDE IN AQUEOUS SOLUTIONS*

Abstract— N-Hydroxypyridine-2-thione, 2-S-PyrNOH, a potent antimicrobial, antifungal and anticancer agent, is photochemically active and upon UV irradiation generates free radicals. We have employed EPR and the spin-trap 5,5-dimethyl-l-pyrroline TV-oxide (DMPO) to investigate the photochemistry in aqueous solutions of 2-S-PyrNOH (used here in the form of a sodium salt, 2-S-PyrNONa). We found that upon photoactivation 2-S-PyrNONa can follow two different pathways: it can produce hydroxyl radicals and/or it can act as a photoreducing agent. The capacity of 2-S-PyrNONa to produce “OH” radicals has been demonstrated by: (1) EPR detection of the DMPO/OH adduct in UV-irradiated samples; (2) inhibition of the DMPO/OH formation by OH scavengers such as methyl alcohol, formate and DMSO and (3) by detection of EPR signals of DMPO adducts with radicals derived from reaction of OH with these inhibitors. The photoreductive capacity of 2-S-PyrNONa was deduced from the observation that the amplitude of the EPR signal of the spin adduct DMPO/OH decreased on UV irradiation in air-free pH 7.0 buffers and that the signal recovered in the dark and after aeration. The ability to generate free radicals upon UV irradiation suggests that 2-S-PyrNONa can be regarded as a potential photocytotoxic agent. This feature may be relevant to the biological action of this compound. Our findings also emphasize that caution should be used when 2-S-PyrNOH is employed as a source of OH radicals in biological or chemical systems.     

UV-generated free radicals (FR) in skin: their prevention by sunscreens and their induction by self-tanning agents

In the past few years, the cellular effects of ultraviolet (UV) irradiation induced in skin have become increasingly recognized. Indeed, it is now well known that UV irradiation induces structural and cellular changes in all the compartments of skin tissue. The generation of reactive oxygen species (ROS) is the first and immediate consequence of UV exposure and therefore the quantitative determination of free radical reactions in the skin during UV radiation is of primary importance for the understanding of dermatological photodamage. The RSF method (radical sun protection factor) herein presented, based on electron spin resonance spectroscopy (ESR), enables the measurement of free radical reactions in skin biopsies directly during UV radiation. The amount of free radicals varies with UV doses and can be standardized by varying UV irradiance or exposure time. The RSF method allows the determination of the protective effect of UV filters and sunscreens as well as the radical induction capacity of self-tanning agents as dihydroxyacetone (DHA). The reaction of the reducing sugars used in self-tanning products and amino acids in the skin layer (Maillard reaction) leads to the formation of Amadori products that generate free radicals during UV irradiation. Using the RSF method three different self-tanning agents were analyzed and it was found, that in DHA-treated skin more than 180% additional radicals were generated during sun exposure with respect to untreated skin. For this reason the exposure duration in the sun must be shortened when self-tanners are used and photoaging processes are accelerated.     

Singlet oxygen-mediated protein oxidation: evidence for the formation of reactive side chain peroxides on tyrosine residues

Singlet oxygen (1O2) is generated by a number of enzymes as well as by UV or visible light in the presence of a sensitizer and has been proposed as a damaging agent in a number of pathologies including cataract, sunburn, and skin cancers. Proteins, and Cys, Met, Trp, Tyr and His side chains in particular, are major targets for 1O2 as a result of their abundance and high rate constants for reaction. In this study it is shown that long-lived peroxides are formed on free Tyr, Tyr residues in peptides and proteins, and model compounds on exposure to 1O2 generated by both photochemical and chemical methods. The yield of these species is significantly enhanced in D2O and decreased by azide. Nuclear magnetic resonance and mass spectroscopic analysis of reaction mixtures, or materials separated by high-performance liquid chromatography, are consistent with the initial formation of an (undetected) endoperoxide that undergoes rapid ring-opening to give a hydroperoxide situated at the C1 ring-position (i.e. para to the phenolic group). In the presence of a free alpha-amino group (e.g. with free Tyr), rapid ring-closure occurs to give an indolic hydroperoxide that decays into the corresponding alcohol, 3a-hydroxy-6-oxo-2,3,3a,6,7,7a-hexahydro-1H-indole-2-carboxylic acid. Hydroperoxides that lack a free alpha-amino group (e.g. those formed on 3-(4-hydroxyphenyl)propionic acid, N-Ac-Tyr and Tyr-containing peptides) are longer-lived, with half-lives of hours to days. These species undergo slow decay at low temperatures to give the corresponding alcohol. Their rate of decay is enhanced at 37 degrees C, or on exposure to UV light or metal ions, and gives rise to reactive radicals, via cleavage of the peroxide bond. These radicals have been characterized by electron paramagnetic resonance spin trapping. These studies demonstrate that long-lived Tyr-derived peroxides are formed on proteins exposed to 1O2 and that these may promote damage to other targets via further radical generation.     

PHOTOAGING EFFECTS ON SPECTRAL TRANSMITTANCE OF PLASTIC FILTERS

We examined the effects of ultraviolet (UV) radiation in combination with high levels of infrared (IR) radiation on the spectral transmittance of plastic filters. The biological action spectrum for damage to the human eye and skin changes dramatically in the 300-400 nm wavelength range. Cut-off filters used in this region to shape the spectrum of exposure sources are thus critical to the design of experiments which use broadband light sources. The changes in transmittance of three types of plastic filters were observed over an exposure period of 1000 h. One set of three filters was exposed mainly to UV radiation, while the other set was exposed to UV radiation plus IR radiation. Filters exposed to both UV and IR radiation showed spectral changes in their transmittance, while the filters exposed to UV only showed no measurable changes.     

Radiation-induced electron paramagnetic resonance signal and soybean isoflavones content

Electron Paramagnetic Resonance (EPR) is a well-known spectroscopic technique that detects paramagnetic centers and can detect free radicals with high sensitivity. In food, free radicals can be generated by several commonly used industrial processes, such as radiosterilization or heat treatment. EPR spectroscopy is used to detect radioinduced free radicals in food. In this work the relation between EPR signal induced by gamma irradiation treatment and soybean isoflavones content was investigated. Present results did not show correlation between total isoflavones content and the EPR signal. Nevertheless, some isoflavone contents had a negative correlation with the radiation-induced EPR signal.     

Divergent optimum levels of lycopene,beta-carotene and lutein protecting against UVB irradiation in human fibroblastst

Exposure of living organisms to UV light leads to photooxidative reactions. Peroxyl radicals are involved in the propagation of lipid peroxidation. Carotenoids are dietary antioxidants and show photoprotective effects in human skin, efficiently scavenging peroxyl radicals and inhibiting lipid peroxidation. Cultured human skin fibroblasts were used to examine the protective effects of the carotenoids, lycopene, beta-carotene and lutein on UVB-induced lipid peroxidation. The carotenoids were delivered to the cells using liposomes as the vehicle. The cells were exposed to UVB light for 20 min. Lycopene, beta-carotene and lutein were capable of decreasing UV-induced formation of thiobarbituric acid-reactive substances at 1 h to levels 40-50% of controls free of carotenoids. The amounts of carotenoid needed for optimal protection were divergent at 0.05, 0.40 and 0.30 nmol/mg protein for lycopene, beta-carotene and lutein, respectively. Beyond the optimum levels, further increases of carotenoid levels in cells led to prooxidant effects.     

PHOTODESTRUCTION OF PHAEOMELANIN

Abstract. Phaeomelanin readily decomposes by the action of UV light and oxygen. This finding may explain many of the abnormal reactions of the skin of redheads and blondes to sunlight, including their high susceptibility to skin cancer. In aqueous solution, phaeomelanin displays an EPR signal rich in hyperfine splitting, consistent with structural information.  

SUMMARY

Phaeomelanin readily, decomposes by the action of UV light and oxygen. This finding may explain many of the abnormal reactions of the skin of redheads and blondes to sunlight, including their high susceptibility to skin cancer. We have yet to identify any of the fragmentation products. Chromatographic analysis of the photolysate indicates at least 17 different compounds. Work on the reactions of phaeomelanin with superoxide, and the identification, synthesis, and physiological evaluation of these photoproducts as etiological factors in sunburn and carcinogenesis is in progress. In aqueous solutions, phaeomelanin displays an air stable EPR signal rich in hyperfine splitting. Complete analysis of this signal will provide a better understanding of the structure of the pigment.     

Ruby Laser Irradiation (694 nm) of Human Skin Biopsies: Assessment by Electron Spin Resonance Spectroscopy of Free Radical Production and Oxidative Stress during Laser Depilation

Human skin biopsies (hair-bearing scalp skin and non-hair-bearing breast skin) were treated with t-butylhydroperoxide, irradiated with UV light (UVR) or irradiated with 694 nm ruby laser red light. Free-radical production and oxidative stress were assessed with electron spin resonance spectroscopy (ESR) using the ascorbate radical as a marker. In comparison with both UVR and t-butyl-hydroperoxide (which readily induce the ascorbate radical in hair-bearing and hairless skin), 694 nm red light does not result in the formation of the ascorbate radical in detectable concentrations. Spin-trapping experiments with the spin trap 5,5-dimethyl-1-pyrroline N-oxide (DMPO) showed that while free radicals could be detected after treatment of skin with t-butylhydroperoxide, radicals could not be trapped after laser treatment. Treatment of lasered skin (containing DMPO) with t-butylhydroperoxide produced radical adducts as well as the ascorbate radical, demonstrating that the laser neither depletes endogenous ascorbate nor the preadministered spin trap. It is concluded that 694 nm red light does not induce oxidative stress in human skin in levels comparable either to t-butyl hydroperoxide or UV light.     

UV-induced free radicals in oriented DNA.

Abstract— Samples of oriented DNA containing 30% water were UV-irradiated at 77 K and investigated by electron paramagnetic resonance (EPR). The EPR spectra recorded in directions parallel and perpendicular to the DNA fibre direction showed that at low UV doses the induced free radicals are very similar to those induced by γ-irradiation at the same temperature. The γ-induced free radicals have previously been analysed and found to consist mainly of anionic free radicals on thymine and cationic free radicals on guanine. At higher UV doses or by suitable annealing of the samples given a low UV dose, significant amounts of hydrogen-addition radicals on thymine were observed. The quantum yield of free radical formation for irradiation at 300nm ± 10nm was estimated to 10^--⁴. We also made a quantitative determination of the UV-induced free radicals inside an optically effective volume of the sample. The following free radical induction frequencies at 77 K were estimated: γ-rays: 2 × 10^--¹² free radicals per rad per dalton and UV (300nm): 6 × 10^--¹² free radicals per J/m² per dalton.     

Dosimetric characteristics of different types of saccharides: An EPR and UV spectrometric study

The time stability and dose response of the free radicals produced in various types of “less-studied” mono- and disaccharides by γ-radiation is studied by EPR (Electron Paramagnetic Resonance) and UV spectrometry. The time evolution of the shape of the EPR spectra of irradiated saccharides is investigated from 5 min to 5 months after irradiation. The intensity of the stable EPR signal is studied as a function of the absorbed γ-dose in the range 0.5–20 kGy. Aqueous solutions of irradiated solid saccharides exhibit a UV absorption maximum in the range 250–290 nm. A linear dependency is found between the magnitude of the UV absorption maximum and the absorbed γ-dose. The time stability of the UV absorption maximum is also studied for every saccharide. The results are compared with those obtained for irradiated sucrose.     

Radical protection by sunscreens in the infrared spectral range

One essential reason for skin ageing is the formation of free radicals by excessive or unprotected sun exposure. Recently, free radical generation in skin has been shown to appear not only after irradiation in the UV wavelength range but also in the infrared (IR) spectral range. Sunscreens are known to protect against radicals generated by UV radiation; however, no data exist for those generated by IR radiation. This paper has investigated four different, commercially available sunscreens and one COLIPA standard with regard to radical formation in the skin after IR irradiation, using electron paramagnetic resonance spectroscopy. The use of sunscreens has led to reduced amounts of radicals compared to untreated skin. Furthermore, absorption and scattering properties and the radical protection factor of the formulations were determined to investigate their influence on the radical protection of the skin. None of these formulations contained an optical absorber in the IR range. The protection efficiency of the sunscreens was shown as being induced by the high scattering properties of the sunscreens, as well as the antioxidants contained in the formulations.