Time-resolved detection of melanin free radicals quenching reactive oxygen species

Melanin, a ubiquitous, heterogeneous biological polymer composed of many different monomers, contains a population of stationary, intrinsic semiquinone-like radicals. Additional extrinsic semiquinone-like radicals are reversibly photogenerated with visible or UV irradiation. The free radical chemistry of melanin is complex and not well characterized, especially the photochemistry of melanin in the presence of oxygen. To determine directly how melanin reacts in the presence of oxygen, time-resolved electron paramagnetic resonance (TREPR) spectroscopy was used to examine melanin free radical chemistry in human retinal pigment epithelium (RPE) cells under aerobic and anaerobic conditions. A TREPR difference spectrum was used to explore the nature of melanin chemistry in the presence of oxygen. The position and symmetrical line shape of the TREPR three-dimensional difference spectrum shows that when reactive oxygen species (ROS) are scavenged, only one of the two or more chemically different melanin free radical species participates in ROS scavenging. This protective melanin radical species exists in both the extrinsic and intrinsic populations of melanin free radicals, allowing melanin to protect the RPE from toxic species in both the light and dark.     

Effects of photodegradation on the physical and antioxidant properties of melanosomes isolated from retinal pigment epithelium

Melanosomes of the retinal pigment epithelium (RPE) are relatively long-lived organelles that are theoretically susceptible to changes induced by exposure to visible light. Here melanosomes were isolated from porcine RPE cells and subjected to high intensity visible light to determine the effects of illumination on melanosome structure and on the content and antioxidant properties of melanin. As compared to untreated melanosomes, illuminated granules showed morphologic changes consistent with photodegradation, which included variable reductions in electron density demonstrated by transmission electron microscopy (TEM), and particle fragmentation and surface disruption revealed by scanning electron microscopy (SEM) and atomic force microscopy. Illuminated melanosomes had lower melanin content, indicated by measures of absorbance and electron spin resonance (ESR) signal intensity, and reduced ability to bind iron, shown by chemical and ESR analyses. Compared to untreated melanosomes, ESR-spin trapping analyses further indicated that illuminated melanosomes show increased photogeneration of superoxide anion and reduced ability to inhibit the iron ion-catalyzed free radical decomposition of hydrogen peroxide. It appears therefore that visible light irradiation can disrupt the structure of RPE melanosomes and reduce the amount and antioxidant properties of melanin. Some of these changes occur in human RPE melanosomes with aging and the results obtained here suggest that visible light irradiation is at least partly responsible. The consequence of light-induced changes in RPE melanosomes may be a diminished capacity of melanin to help protect aged cells from oxidative damage, perhaps increasing the risk of diseases with an oxidative stress component such as age-related macular degeneration.     

15N CIDNP investigations of the peroxynitric acid nitration of L-tyrosine and of related compounds

Peroxynitric acid (O2NOOH) nitrates L-tyrosine and related compounds at pH 2-5. During reaction with O2(15)NOOH in the probe of a 15N NMR spectrometer, the NMR signals of the nitration products of L-tyrosine, N-acetyl-L-tyrosine, 4-fluorophenol and 4-methoxyphenylacetic acid appear in emission indicating a nitration via free radicals. Nuclear polarizations are built up in radical pairs [15NO2* , PhO*]F or [15NO2* , ArH*+]F formed by diffusive encounters of 15NO2 with phenoxyl-type radicals PhO or with aromatic radical cations ArH*+. Quantitative 15N CIDNP investigations with N-acetyl-L-tyrosine and 4-fluorophenol show that the radical-dependent nitration is the only reaction pathway. During the nitration reaction, the 15N NMR signal of 15NO3- also appears in emission. This is explained by singlet-triplet transitions in radical pairs [15NO2* , 15NO3*]S generated by electron transfer between O2(15)NOOH and H15NO2 formed as a reaction intermediate. During reaction of peroxynitric acid with ascorbic acid, 15N CIDNP is again observed in the 15N NMR signal of 15NO3- showing that ascorbic acid is oxidized by free radicals. In contrast to this, O2(15)NOOH reacts with glutathione and cysteine without the appearance of 15N CIDNP, indicating a direct oxidation without participation of free radicals.     

中性溶液中抗坏血酸在六氰合亚铁酸钴铜膜修饰铂电极上现场红外光谱电化学研究

抗坏血酸 ( AA)是生物化学和生物医学中的一种重要生理物质 ,因而在电催化和电化学传感器领域得到了广泛的研究 .对抗坏血酸的研究主要集中在两个方面 :一方面是研究 AA在各种电极上的电氧化机理、动力学参数及电催化机理 [1,2 ] ;另一方面是为了监测各种生物化学和生物医学中 AA的浓度发展起来的 AA传感器 .利用碳电极的各种预处理方法 [3,4 ]以及利用各种化学修饰电极 [5,6 ]来促进AA的电氧化动力学过程及解决 AA与各种共存物如多巴胺及脲酸等之间的相互干扰问题 .六氰合亚铁酸钴铜修饰电极 ( Co Cu HCF/Pt) [7] 在中性溶液中十分…     

ELECTRON SPIN RESONANCE OF MELANIN FROM HAIR. EFFECTS OF TEMPERATURE, pH AND LIGHT IRRADIATION

Abstract— The variation with temperature, pH and light of the ESR signal of hydrated melanin powders from Japanese black hair has been studied. An explanation of the results is proposed on the basis of quinhydrone type complexes and of acid-base equilibria of melanin and its semiquinone radicals. During exposure to light of wavelengths 254–600 nm, both stable and unstable radicals have been observed. The action spectrum for the formation of stable melanin radicals has been determined.     

Effect of ascorbic acid (vitamin C) on the ESR spectra of the red and black hair: pheomelanin free radicals are not always present in red hair

Increased incidence of melanoma in the population with red hair is conditioned by synthesis of pheomelanin pigments in the skin and their phototoxic properties. The recent research has shown that free radicals of pheomelanin are produced not only by the influence of UV irradiation, but also in UV‐independent pathways of oxidative stress. It has been ascertained, that the color of the hair is not always determinant of the amount of pheolemanin radicals in red hair. Therefore, in order to evaluate the risk of melanoma in different individuals, it is necessary to define the amount of free radicals of pheomelanin in red hair using ESR spectroscopy method. Besides, it is very important to find effective antioxidant, capable of neutralizing free radicals of pheomelanin. It was proved that ascorbic acid neutralizes free radicals of pheomelanin very effectively. The main goal of our research was to define the presumably optimal concentration of ascorbic acid as an antioxidant and study the kinetics of the influence of this concentration on red and black hair. It has been found out, that ascorbic acid influences the free radicals of red and black hair, and its appropriate optimal concentration is 10 mM. The obtained results can be considered in dermatology and cosmetology. Copyright © 2015 John Wiley & Sons, Ltd.     

Characterization of Retinal Pigment Epithelial Melanin and Degraded Synthetic Melanin Using Mass Spectrometry and In Vitro Biochemical Diagnostics

With increasing age, there is an observable loss of melanin in retinal pigment epithelial (RPE) cells. It is possible that degradation of the pigment contributes to the pathogenesis of retinal disease, as the cellular antioxidant material is depleted. Functionally, intact melanin maintains protective qualities, while oxidative degradation of melanin promotes reactive oxygen species (ROS) generation and formation of metabolic byproducts, such as melanolipofuscin. Understanding the structural and functional changes to RPE melanin with increasing age may contribute to a better understanding of disease progression and risk factors for conditions such as age‐related macular degeneration (AMD). In this study, human donor RPE melanin is characterized using MALDI mass spectrometry to follow melanin degradation trends. In vitro models using ARPE‐19 cells are used to assess photo‐reactivity in repigmented cells. Significant protection against intracellular ROS produced by blue light is observed in calf melanin‐pigmented cells versus unpigmented and black latex bead controls (P < 0.0001). UV‐B exposure to aged human melanin‐pigmented cells results in a significant increase in nitric oxide production versus control cells (P < 0.001). Peroxide‐treated synthetic melanin is characterized to elucidate degradation products that may contribute to RPE cell damage.     

Photobleaching of melanosomes from retinal pigment epithelium: II. Effects on the response of living cells to photic stress

Melanosomes of the retinal pigment epithelium (RPE) are long lived organelles that may undergo photobleaching with aging, which can diminish the antioxidant efficiency of melanin. Here, isolated porcine RPE melanosomes were experimentally photobleached with visible light to simulate aging and compared with untreated granules or control particles (black latex beads) for their effects on the survival of photically stressed ARPE-19 cultures. Particles were delivered to cultures for uptake by phagocytosis then cells were exposed to violet light and analyzed by a new live cell imaging method to identify the time of apoptotic blebbing as a dynamic measure of reduced cell survival. Results indicated that untreated melanosomes did not decrease photic injury to ARPE-19 cells when compared with cells lacking particles or with cells containing control particles, as might be expected if melanin performed an antioxidant function. Instead cells with untreated melanosomes showed reduced survival indicated by an earlier onset of blebbing and a lower fraction of surviving cells after photic stress. Cell survival was reduced even further in stressed cells containing melanosomes that were photobleached, and survival decreased with increasing photobleaching time. Photobleaching of RPE melanosomes therefore makes cells containing them more sensitive to light-induced cytotoxicity. This observation raises the possibility that aged melanosomes increase RPE cell photic stress in situ, perhaps contributing to reduced tissue function and to degeneration of the adjacent retina that the RPE supports. How melanosomes (photobleached or not) interact with their local subcellular environment to modify RPE cell survival is poorly understood and is likely determined by the physicochemical state of the granule and its constituent melanin. The live cell imaging method introduced here, which permitted detection of a graded effect of photobleaching, provides a sensitive bioassay for probing the effects of melanosome modifications.     

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.     

Photoprotection by porcine eumelanin against singlet oxygen production

Melanin, a major pigment found in retinal pigment epithelium (RPE) cells, is considered to function in dual roles, one protective and one destructive. By quenching free radical species and reactive oxygen species (ROS) melanin counteracts harmful redox stress. However, melanin is also thought to be capable of creating ROS. In this destructive role, melanin increases redox strain in the cell. This study uses readily available eumelanin extracted from porcine RPE cells as a more authentic model than synthetic melanin to determine specific mechanisms of melanin activity with regard to singlet oxygen in the presence and absence of rose bengal, a singlet-oxygen photosensitizer. Optical detection of singlet-oxygen was determined by monitoring the bleaching of p-nitrosodimethylaniline in the presence of histidine. Production of singlet oxygen in aqueous oxygen-saturated solutions of rose bengal without eumelanin was readily accomplished. In contrast, detection of singlet oxygen in oxygen-saturated solutions of eumelanin without rose bengal failed, consistent with results of others. However, a significant decrease in singlet oxygen production by rose bengal was observed in the presence of eumelanin. After correction for light absorption and chemical bleaching of eumelanin, the results show that eumelanin also provides a photoprotective mode arising from chemistry, that is, not just the physical process of light absorption followed by energy dissipation as heat.     

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.     

Antioxidant properties of melanin in retinal pigment epithelial cells

The retinal pigment epithelium (RPE) is a monolayer of highly pigmented cells lining the inner aspect of Bruch's membrane. This pigmentation is due to eumelanin and a possible antioxidant role of melanin is reported here. The photo-oxidation of A2E, a constituent of RPE lipofuscin, leads to the sequential addition of up to nine oxygen atoms and/or the addition or loss of two hydrogen atoms. These photo-oxidations were investigated in the presence and absence of either calf or human RPE melanin in A2E-laden RPE cells. It was found that calf melanin was protective against the photo-oxidation of A2E, with an inhibition of oxidation of up to 50% in the case of the addition of two oxygen atoms. Calf melanin was also protective against blue light-induced damage to RPE cells. In addition this ability appears to decrease in humans as they grow older. With aging, a melanin-lipofuscin complex called melanolipofuscin forms. It is suggested that the oxidation or photo-oxidation of A2E in vivo may contribute to the age-related deterioration of the anti-oxidant role of RPE melanin and lead to various retinal disorders, such as age-related macular degeneration.     

Ascorbic Acid Determination by Hydrophobic Liquid Chromatography of the Osazone Derivative. Application to the Analysis of Aqueous Humor

Abstract

The dinitrophenylhydrazine colorimetric method for the analysis of ascorbic acid has been converted to a high-performance liquid chromatography (HPLC) procedure. The bis-(dinitrophenyl) hydrazone resulting from the reaction of ascorbic acid and dinitrophenylhydrazine is separated on a reversed-phase system from other components in the reaction mixture and used for quantitation purposes. The specificity of the analysis is thus greatly improved and its sensitivity is brought down to the picomol level. The HPLC procedure described here is therefore specially suitable for the analysis of very small volume samples when high specificity and sensitivity are of prime importance. With this method, as in the original colorimetric procedure, it is also possible to specify the contribution of the reduced and oxidized forms of ascorbic acid to the total amount present in the sample. Basically, the same approach could be advantageously used to improve other colorimetric methods of analysis. Human aqueous humor samples taken from senile cataract patients at the time of surgery show variable but significant amounts of oxidized ascorbic acid. This oxidized traction, therefore, should not be neglected as is frequently done when the ascorbic acid system of the aqueous humor is studied.     

Antioxidant activity of papaya seed extracts

The antioxidant activities of the ethanol, petroleum ether, ethyl acetate, n-butanol and water extract fractions from the seeds of papaya were evaluated in this study. The ethyl acetate fraction showed the strongest DPPH and hydroxyl free radical-scavenging activities, and its activities were stronger than those of ascorbic acid and sodium benzoate, respectively. The n-butanol fraction demonstrated the greatest ABTS? radicals scavenging activity. The ethyl acetate fraction and the n-butanol fraction not only showed higher antioxidant activities than the petroleum ether fraction, water fraction and ethanol fraction, but also showed higher superoxide anion and hydrogen peroxide radicals scavenging activities than those of the other extract fractions. The high amount of total phenolics and total flavonoids in the ethyl acetate and n-butanol fractions contributed to their antioxidant activities. The ethyl acetate fraction was subjected to column chromatography, to yield two phenolic compounds, p-hydroxybenzoic acid and vanillic acid, which possessed significant antioxidant activities. Therefore, the seeds of papaya and these compounds might be used as natural antioxidants.     

Characterization of human hair melanin and its degradation products by means of magnetic resonance techniques

Melanin granules (MGs) have been extracted from human Chinese black hairs by either acid hydrolysis (CH-type MGs) or enzymatic digestion (CP-type MGs), and their chemical structure investigated at the solid state by means of (13)C cross polarization magic angle spinning (CPMAS NMR) and EPR spectroscopy. Both types of MGs contain a large amount of protein that is tightly bound to the true melanin polymer, with CP-type MGs having a larger protein content than CH-type ones. Moreover, MGs may also contain variable amounts of lipid-like material. A high amount of paramagnetic metals is detected by EPR in CP-type MGs, in particular Fe(III). Iron can be bound in two chemical forms: as isolated high spin Fe(III) ions with rhombic symmetry and as small oxy-hydroxy Fe(III) aggregates. Iron is poorly available to chelators. CH-type MGs contain much fewer metals. CP-type MGs have then been subjected to partial bleaching by hydrogen peroxide in ammonia, yielding a residual solid, called residual oxidized melanin (ROM) and a soluble but still pigmented fraction called melanin free acid (MFA). MFA can be isolated by precipitation at acidic pH. The (13)C-CPMAS NMR and EPR spectra of these derivatives indicated that ROM has a structure very similar to that of parent MGs, whereas MFA shows a decrease of the protein content with respect to the melanin and a decreased amount of bound iron. Thus, the oxidative degradation of CP-type MGs is a process not involving the bulk of MGs, but rather it proceeds from the solvent-exposed outer parts to the interior.     

ESR STUDY OF A NEW LIGHT-SENSITIVE FREE RADICAL IN THE COATS OF WHEAT AND OTHER SEEDS

Abstract— The endogenous free radical signal in the coat of wheat and other seeds is found to increase rapidly upon exposure to light from a high pressure mercury lamp reaching a saturation value in about one hour. Upon dark storage the signal slowly decays to approximately its initial value after a number of days. Re-exposure to the light causes the signal to again increase. The signal also depends inversely and reversibly upon moisture content. The dynamic behavior of the signal indicates that the radicals are produced from a limited number of centers in the seed coat. The identity of these centers is still unknown, but our results rule out such possibilities as chlorophyll, melanin and phenolic compounds and suggest that a new light-sensitive component is present in the outer coating of seeds.     

The pro-oxidant effects of interactions of ascorbate with photoexcited melanin fade away with aging of the retina

Photoexcited melanin from retinal pigment epithelium (RPE) has been shown to induce photo-oxidation of ascorbate with concomitant generation of hydrogen peroxide. The aim of this study was to test whether the age-related changes in melanin content and distribution in the RPE affect the susceptibility of RPE cells to ascorbate-mediated photo-oxidation. Our results demonstrate that there is an age-dependent shift in the pathways with which ascorbate interacts in human RPE. In young RPE, melanin-ascorbate interactions may lead to pro-oxidant effects, but in the aged there is no net increase in photo-oxidation in the presence of ascorbate in comparison with samples without ascorbate. However, as ascorbate undergoes light-induced depletion and photogenerates ascorbyl free radical in the old RPE cells with initial yields similar to that observed for young RPE, an influence of ascorbate on oxidation pathways is revealed in the old RPE as well. Interestingly, the pro-oxidant effects of photoexcited melanolipofuscin-ascorbate interactions are greater than for photoexcited melanosomes when normalized to the same melanin content. The pro-oxidant effects of photoexcited melanin-ascorbate interactions are strongly dependent on the irradiation wavelength, this being the greatest for the shortest wavelength studied (340 nm) and steeply decreasing with increasing wavelength but still detectable even at 600 nm.     

INDUCTION OF FREE RADICALS IN DNA BY PROFLAVINE AND VISIBLE LIGHT: INFLUENCE OF OXYGEN AND IONIC STRENGTH

Abstract —The photosensitization of native DNA is observed as an induction of free radicals in the DNA moiety of proflavine-DNA complexes. The intensity of the electron paramagnetic resonance spectra (at 77 K) is a measure of the number of free radicals present in frozen solutions of DNA-proflavine complexes after irradiation with visible light (Λ > 320 nm). In the absence of O₂, the photosensitization is significant but very low; it increases slightly with increasing NaCl ionic strength; it appears to be due to intercalated dye molecules and the qualitative analysis of the spectra obtained shows that mainly thymidine is involved. The reaction measured after saturation with O₂ is the same as the reaction in air but is quantitatively higher; the free radicals observed are peroxides. This induction of free radicals appears to be due to the intercalated dye molecules, each molecule acting independently. The important observation is a very sharp and large (around a hundred-fold) increase in the photosensitizing efficiency of the bound dye molecules occurring in NaCl between μ, # 0–25 and μ= 0–5 and in MgCl₂ between μ# 0–01 and μ=0–1.     

ACUTE EFFECTS OF NEAR ULTRAVIOLET AND VISIBLE LIGHT ON THE CUTANEOUS ANTIOXIDANT DEFENSE SYSTEM

Reactive oxygen species are considered to play an important role in cutaneous pathology. Enzymic and non-enzymic antioxidants can prevent oxidative damage but may be overcome by strong pro-oxidative stimuli. The acute effect of a single exposure to near ultraviolet (UVA)/visible radiation (greater than 320 nm) on various skin antioxidants was examined in hairless mice immediately after irradiation. Impairment of cutaneous catalase and glutathione reductase activity was observed. Superoxide dismutase and glutathione peroxidase were not significantly influenced. Inhibition of catalase may render skin more susceptible to the damaging effects of hydrogen peroxide and its reaction products such as the hydroxyl radical. Partially diminished glutathione reductase activity is not accompanied by a change in reduced/oxidized glutathione level immediately after irradiation. There was a tendential (not statistically significant) decrease in cutaneous tocopherol, ubiquinol + ubiquinone 9 and ascorbic acid levels, either indicating direct photodestruction or consumption by reaction products of photooxidative stress. This partial impairment of the cutaneous antioxidant defense system by near ultraviolet/visible light, showing that the most susceptible component in skin is catalase, suggests possible pharmacological interventions.     

Electrospray mass spectrometric analysis of 5-hydroperoxy and 5-hydroxyeicosatetraenoic acids generated by lipid peroxidation of red blood cell ghost phospholipids

Recent evidence suggests that generation of hydroxyl radicals in the presence of lipid membranes can lead to oxidation of arachidonic acid esterified to glycerophospholipids and the production of compounds isomeric to prostaglandins, thromboxanes, and leukotrienes. Liquid chromatography tandem mass spectrometry and multiple reaction monitoring were employed to quantitate the production of 5-hydroxyeicosatetraenoic acid (5-HETE), 5-hydroperoxyeicosatetraenoic acid (5-HPETE), and 5-oxo-eicosatetraenoic acid (5-oxo-ETE) in red blood cells ghosts treated with t-butylhydroperoxide (tBuOOH). Untreated red blood cell ghosts were found to contain low, but measurable quantities of these three 5-oxygenated eicosanoids as phospholipid esters. Following treatment, there was approximately a 53- and 22. 5-fold increase in 5-HETE and 5-HPETE, respectively, and an 8. 5-fold increase in 5-oxo-ETE. The formation of these compounds was inhibited nearly 90% by the antioxidants butylated hydroxytoluene, ascorbic acid, and resveratrol providing further evidence for free radical mediated oxidation of arachidonic acid. This analytical protocol provided sufficient sensitivity for detection of these compounds in studies in which previous analysis by high-pressure liquid chromatography with UV detection failed to detect their presence. These results reveal that the biologically active eicosanoids 5-HETE, 5-HPETE, and 5-oxo-ETE are formed esterified to phospholipids following exposure of cellular membranes to reactive oxygen species and free radicals in a model system where intracellular antioxidant mechanisms were depleted.