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.     

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.     

ESR DETECTION OF ENDOGENOUS ASCORBATE FREE RADICAL IN MOUSE SKIN: ENHANCEMENT OF RADICAL PRODUCTION DURING UV IRRADIATION FOLLOWING TOPICAL APPLICATION OF CHLORPROMAZINE

Using electron spin resonance spectroscopy, we observed that UV radiation (330 nm) increased the endogenous ascorbate free radical concentration in hairless mouse (HRS/J) skin. When the skin was topically treated with a chlorpromazine solution prior to illumination, UV irradiation caused the ascorbate free radical concentration to increase even more. This observation suggests that there is an increased UV-induced oxidative stress in the presence of chlorpromazine, probably caused by the production of free radicals from chlorpromazine.     

Influence of hydroxypropyl-β-cyclodextrin on the photostability and antiradical activity of Trolox

Trolox (6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid), a derivative of vitamin E, can undergo photolysis upon UV irradiation. In the present study the photodegradation kinetics of Trolox were thus investigated in different systems in the absence and in the presence of titanium dioxide, a physical sunscreen present in several cosmetic products, that can act as photocatalyst. In all the considered media Trolox degraded under UVB light following pseudo-zero order kinetics, probably by a mechanism of photooxidation. The rate of Trolox photodegradation was lower in O/W emulsions than in aqueous solution and in gel; furthermore it was significantly influenced by the presence of TiO₂. Aiming to increase Trolox stability, it was complexed with hydroxypropyl-β-cyclodextrin: the inclusion complex was characterized by phase solubility studies, spectrophotometry and differential scanning calorimetry. The irradiation experiments indicated that complexation with hydroxypropyl-β-cyclodextrin was effective in reducing the photodegradation rate of Trolox. Moreover the host molecule favored the uptake of Trolox into the porcine skin, as shown by in vitro permeation studies. Nevertheless the assay of peroxidation, based on the reaction of malondialdehyde with thiobarbituric acid, indicated that the antioxidant activity of Trolox was maintained even after inclusion in cyclodextrin.     

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.     

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.     

Synthesis and ABTS Radical,MMP‐1 Inhibitory Activity of CAPE Analogues

In the photoaging process of skin, the ultraviolet (UV)‐induced reactive oxygen species (ROS) is the key regulator of matrix metalloproteinase (MMPs) expression. In this study, a series of Caffeic acid phenethyl ester (CAPE) analogues were synthesized by conjugating the group VI elements (selenium, sulfur, oxygen)‐containing aliphatic alcohols to polyphenolic acids. Their biological activities were evaluated by in vitro testing of their radical scavenging activity the of ABTS [2,2′‐azinobis‐(3‐ethyl‐benzothiazoline‐6‐sulfonic acid)] radical and inhibitory effect against the matrix metalloproteinase‐1 (MMP‐1) activity of collagen degradation and cytotoxicity of a human dermal fibroblast skin cell. Our results suggest these compounds displayed moderate anti‐free radical, potent MMP‐1 inhibitory, and low cytotoxic activities.     

Unprecedented Acid‐Promoted Polymerization and Gelation of Acrylamide: A Serendipitous Discovery

Dilute acid polymerizes degassed, aqueous acrylamide with concomitant gelation, without the need for added free radical initiator or cross‐linking agent. This reaction is accelerated by sonication or UV irradiation, but inhibited by adventitious oxygen or the addition of a free radical inhibitor, suggesting an acid‐accelerated free radical process. The resulting hydrogels are thixotropic in nature and partially disrupted by the addition of chaotropic agents, indicating the importance of hydrogen bonding to the 3D network. This discovery was made while trying to prepare pectin‐polyacrylamide hydrogels. We observed that pectin initiated the gelation of acrylamide, but only if the aqueous pectin samples had a pH lower than ca. 5.     

THE OXIDATION OF ASCORBATE BY ELECTRON AFFINIC DRUGS AND CARCINOGENS

Abstract— The nitrobenzenes, the carcinogens 4-nitropyridine- N -oxide and 4-nitro-quinoline- N -oxide as well as the nitrofurans, also known to be carcinogenic, have been found to enhance the reaction of ascorbate with oxygen. The reaction results in the oxidation of ascorbate, the production of dehydroascorbate, superoxide radical, peroxide and water. The drugs are not reduced to stable intermediates during the oxidation but are recycled to their original state. The oxygen consumption is partially inhibited by either superoxide dismutase or catalase. If both superoxide dismutase and catalase are included in the reaction mixture, total oxygen consumption was equal to the amount expected for oxidation of ascorbate to dehydroascorbate and reduction of oxygen to water. The oxygen consumption was inhibited by ferricytochrome c. Semiquinones, nitro and hydroxylamine radicals, produced by electron transfer from ascorbate, reduce ferricytochrome c. These oxygen reactive radicals are responsible for the stimulation of oxygen utilization and ascorbate oxidation. In addition we have found that Ehrlich cells, containing catalase and superoxide dismutase, inhibit the drug catalyzed oxidation of ascorbate. The presence of cyanide, known to inhibit catalase and superoxide dismutase, abolished the cell effect for most of the drugs tested.     

Inhibition of Low-Density Lipoprotein Peroxidation by BHA Use: Fluorimetric Assay

Abstract

In the presence of free radicals, low-density lipoproteins (LDL) undergo oxidation that leads to the formation of lipoperoxides, which cause various damaging effects. Starting from the classical oxygen radical absorption capacity (ORAC) method, a modified method was formulated to develop a laboratory model that is able to deliver information about the use of polyphenols as nutrient supplements. The aim of this work was to establish the possible inhibition of LDL peroxidation by two different antioxidants, Trolox and BHA, and to assess if the effect is either of prevention or repair. 2,2,-Azobis(2-methylpropionamidine) dihydrochloride (AAPH) was used as free radical generator, and fluorescein was used as fluorescent probe. It was noticed that the radical scavenging effect decreases in the order BHA to Trolox to LDL itself. Trolox shows a higher efficacy against the free radicals generated by AAPH than against lipoperoxides, whereas BHA is more efficient against lipoperoxides.     

On the complexation of Trolox with methyl-β-cyclodextrin: characterization, molecular modelling and photostabilizing properties

Exposure to UV radiations could reduce the efficiency of some antioxidants like Trolox (6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid), a water-soluble vitamin E analogue largely employed in cosmetic products. Accordingly, in this paper we examined the possibility of increasing the stability of Trolox towards UVB irradiation by its complexation with methyl-β-cyclodextrin. Formation of the inclusion complex was confirmed by solubility diagrams, differential scanning calorimetry (DSC), and diffusion study through hydrophilic membrane. The stability constants and docking results suggested that the complexation phenomenon was related to the pH of the medium. The photodegradation studies were carried out in different topical formulations (gel, O/W emulsion, and W/O/W emulsion) containing Trolox free or complexed with methyl-β-cyclodextrin. Results showed that in all the cases Trolox degraded following pseudo-zero order kinetics. Moreover, the host molecule increased Trolox photostability also in the presence of TiO₂, a physical sunscreen well-known as photocatalyzer.     

Potential involvement of free radical reactions in ultraviolet light-mediated cutaneous damage

Free radicals are chemical species characterized by an odd number of orbital electrons or by pairs of electrons of similar directional spin isolated singly in separate orbitals. Consequently most of these agents are highly reactive and usually exhibit an extremely short half-life, although due to steric and resonance effects some exceptions occur. Some radicals and their precursors, such as the diradical O₂ which exists in the triplet state, represent a critical and essential element of normal metabolism of aerobic organisms where, under normal circumstances, controlled reduction of reactive oxygen species occurs via the cytochrome oxidase or cytochrome P-450 mixed function monooxygenase systems. In addition to reactive oxygen species, organisms may be subjected to a wide-range of other free radicals or their precursors, including those of both exogenous and endogenous origin. Elaborate defense mechanisms have evolved to avoid cellular damage from these highly reactive species. Enzymes, such as the superoxide dismutase, the glutathione peroxidase/reductase system, and catalase; interactions with conjugated diene systems such as those found in melanins, carotenoids, and tocopherols; and direct reduction by sulphydryl compounds, phenols, and purines represent but a few of these natural defense systems. Despite a strong rationale for considering free radicals as pathologic agents, progress in implicating these agents, or their reactions, in pathologic processes has been arduous. The fore-most hurdle to providing definitive evidence for free radical involvement rests with the highly transient nature of these species, hardly reaching measurable levels in vivo and thereby making rigorous testing of the hypothesis extremely difficult. Indeed, free radical damage has been studied, for the most part, by indirect means–usually by measurement of known free radical reaction intermediates and products from which free radical involvement is implied. Nevertheless, free radical formation has been shown to occur in UV-irradiated skin and a considerable body of circumstantial evidence has been amassed that strongly infers that these agents, or reactions initiated by them, are responsible for at least some of the deleterious effects of UV upon skin.     

PHOTOSENSITIZATION BY DRUGS IN SURFACTANT SOLUTIONS

Abstract— Chlorpromazine, promazine, anthracene and furosemide were tested as photosensitizers using 365 nm UV light in micellar solutions of cationic, anionic and nonionic surfactants. In all cases, micelles enhanced the ability of these compounds to photosensitize the oxidation of 2,5-dimethylfuran and the free radical polymerization of acrylamide. pH variation showed that the base form of chlorpromazine and the acid form of furosemide are the principal photosensitizing forms of these compounds. Rate differences between cationic and anionic surfactant media indicate the cation radical to be the major photochemical species formed from chlorpromazine and promazine in micellar media. Photodechlorination of chlorpromazine accounted for a significantly higher reactivity of chlorpromazine over promazine. Anthracene was found to be a very active photosensitizer by the singlet oxygen mechanism but also yielded a small concentration of cation radicals in micellar solution. In its neutral form, furosemide reacted strongly in both photooxidation and photopolymerization systems.
The implications of this study to drug-induced photosensitivity are that (i) free radical reactions may play a major role, and (ii) these sensitizers are more reactive in a hydrophobic environment, suggesting that the cellular membrane or the hydrophobic surfaces of proteins or DNA are more important sites of action in photosensitivity.     

Studies on bio-antioxidants--prooxidation of vitamin E on the autoxidation of linoleic acid in sodium dodecyl sulfate micelles

The reaction of vitamin E (alpha-tocopherol) with linoleic acid containing peroxidized linoleic acid has been studied. No significant reaction was found in ethanol solution, whereas in sodium dodecyl sulfate micelles vitamin E reacted rapidly with peroxidized linoleic acid, and thereby induced the peroxidation of linoleic acid, leading to oxygen absorption. The reaction kinetics was studied in detail by u.v. spectroscopy, HPLC and ESR spectroscopy. It was found that the main product was alpha-tocopherone with alpha-tocopheroxy radical as the reaction intermediate. A mechanism involving two consecutive bimolecular reactions between peroxidized linoleic acid and alpha-tocopherol and between peroxidized linoleic acid and alpha-tocopheroxy radical, with rate constant 2.93 and 6.21 mol/L-1s-1 respectively is proposed. The micellar effect on the reaction is discussed.     

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.     

Protective role of butylated hydroxytoluene and certain carotenoids in photocarcinogenesis

Butylated hydroxytoluene (BHT) and certain carotenoid pigments have been found to inhibit photocarcinogenesis in animal models. In addition, BHT protects against UV-B-induced erythema and UV-B induction of ornithine decarboxylase. Studies on the photoprotective mechanism(s) of BHT suggested that changes in the physico-chemical properties of the keratin of the stratum corneum layer of skin occurred, leading to increases in UV absorption of that tissue. These changes might be exerted via the anti-radical action of BHT that retards oxidation and prevents cross-linking of the keratin chains, resulting in a diminution of UV-B radiation reaching potential target sites. The carotenoids beta-carotene, canthaxanthin and phytoene also inhibit UV-B carcinogenesis. beta-Carotene and canthaxanthin are excellent quenchers of singlet oxygen, and all three pigments can quench free radicals. beta-Carotene and canthaxanthin have been shown to quench singlet oxygen/free radical reactions in the skin of porphyric mice, and these two pigments as well as phytoene have been found to quench excited species formed on irradiation of mouse skin by UV-B.     

Exacerbating effect of vitamin E supplementation on DNA damage induced in cultured human normal fibroblasts by UVA radiation

The effects of vitamin E supplementation were evaluated in cultured human normal fibroblasts exposed to ultraviolet A radiation (320-380 nm) (UVA). Cells were incubated in medium containing alpha-tocopherol, alpha-tocopherol acetate or the synthetic analog Trolox for 24 h prior to UVA exposure. DNA damage in the form of frank breaks and alkali-labile sites, collectively termed single-strand breaks (SSB), was assayed by the technique of single cell gel electrophoresis (comet assay), immediately following irradiation or after different repair periods. The generation of hydrogen peroxide (H2O2) and superoxide ion (O2.-) was measured by flow cytometry through the oxidation of indicators into fluorescent dyes. It was observed that pretreatment of cells with any form of vitamin E resulted in an increased susceptibility to the photoinduction of DNA SSB and in a longer persistence of damage, whereas no significant change was observed in the production of H2O2 and O2.- reactive oxygen species, compared to untreated controls. These findings indicate that in human normal fibroblasts, exogenously added vitamin E exerts a promoting activity on DNA damage upon UVA irradiation and might lead to increased cytotoxic and mutagenic risks.     

The β-carotene dilemma: ESR study with coordinated peroxyl radicals

The apparently unpredictable behaviour of β-carotene in the supplementation of the diet of smokers is discussed in the light of the reactions of peroxyl radicals with β-carotene in the absence of oxygen. The decay of tert-butylperoxyl radicals in the presence of β-carotene was studied at ambient temperature in non-polar solvents by ESR spectroscopy. The primary reaction in the absence of oxygen is interpreted as a spin-trapping effect of a peroxyl radical by β-carotene producing an intermediate labile free radical, which disappears after recombination with a second tert-butylperoxyl radical. The result is the transformation of β-carotene to a diamagnetic compound with two peroxy bonds. In the presence of chelating transition metals with unpaired d-electrons as electron donors the peroxy group of the oxidized β-carotene can be split to alkoxyl free radicals. The primary attack of tert-butylperoxyl radicals is completely inhibited in the presence of vitamin E followed by production of free aryloxy radicals and the presence of oxygen has no significant effect on this reaction. Spin-trapping of peroxyl radicals by the double bond of vitamin A leads to its oxidation in the absence of vitamin E. Transition metal ions such as Co, Cr, Fe, and Mn, known to be present in the aerosol of cigarette smoke, homolyse the peroxyl bonds of peroxidised β-carotene, which results in cell damage.     

Identification of oxidative processes during simulated mastication of uncooked foods using electron paramagnetic resonance spectroscopy

Electron paramagnetic resonance (EPR) spectroscopy is used to measure directly the generation of free radicals during a simulation of the mastication process. This involves the gentle grinding of the food product in the presence of a spin trap, a molecule which reacts selectively with unstable free radicals to generate (more) stable radical adducts, which can then be characterised. With mushrooms of the Agaricus family, adducts consistent with a carbon-centred radical are seen with a wide range of spin traps and this radical has been confirmed as 4-(hydroxymethyl)phenyl. In plant tissues that are rich in ascorbic acid, this molecule competes successfully with spin traps for the free radicals and the (monodehydro)ascorbate radical, formed by the 1-electron oxidation of ascorbic acid, is seen in the EPR spectra. However, with >50% of the plant tissue samples studied in the present experiment, free radicals resulting from oxidation of the spin traps were observed. The formation of such molecules, for which oxygen was found to be necessary, requires the existence of powerful oxidation processes as the plant tissue is broken down. Such pro-oxidant behaviour is contrary to the popular assumption that the beneficial effects of uncooked plant tissues are the result of their high levels of anti-oxidant molecules.     

Kinetics of oxidation of bilirubin and its protein complex by hydrogen peroxide in aqueous solutions

A comparative study of oxidation reactions of bilirubin and its complex with albumin was carried out in aqueous solutions under the action of hydrogen peroxide and molecular oxygen at different pH values. Free radical oxidation of the pigment in both free and bound forms at pH 7.4 was shown not to lead to the formation of biliverdin, but to be associated with the decomposition of the tetrapyrrole chromophore into monopyrrolic products. The effective and true rate constants of the reactions under study were determined. It was assumed that one possible mechanism of the oxidation reaction is associated with the interaction of peroxyl radicals and protons of the NH groups of bilirubin molecules at the limiting stage with the formation of a highly reactive radical intermediate. The binding of bilirubin with albumin was found to result in a considerable reduction in the rate of the oxidation reaction associated with the kinetic manifestation of the protein protection effect. It was found that the autoxidation of bilirubin by molecular oxygen with the formation of biliverdin at the intermediate stage can be observed with an increase in the pH of solutions.