Approaches to Increasing Skin Melanin with MSH Analogs and Synthetic Melanins

Increased public awareness of ultraviolet (UV) radiation‐induced skin cancers has lead to new interest in technologies for protection from sun exposure. Although many sun protection formulations are available, few of them attempt to achieve that provided by melanin itself, i.e., wide‐spectrum absorbance of radiant energy coupled with anti‐oxidant activity from a single product. In that regard, technologies in two separate areas are at or near the commercialization stage: 1) hormonal enhancement of natural skin melanin content, and 2) inclusion of natural and synthetic melanins in cosmetic formulations to impart melanin‐like color to the skin. In this article, these approaches are briefly summarized using as examples Melanotans I and II®, superpotent analogs of the melanin‐stimulating hormone melanocortin (MSH), and Melasyn®, a group of plant‐derived synthetic melanins that have successfully been incorporated into cosmetic formulations for use as sun protectants and as cover‐ups for problems resulting from uneven pigmentation, such as seen in vitiligo.     

Electron spin relaxation of synthetic melanin and melanin-containing human tissues as studied by electron spin echo and electron spin resonance

Electron spin lattice relaxation times (T1) and the phase memory times (Tm) were obtained for the synthetic melanin system from 3-hydroxytyrosine (dopa) by means of electron spin echo spectroscopy at 77 degrees K. Saturation behavior of the ESR spectra of melanins in melanin-containing tissue and of the synthetic melanin was also determined at the same temperature. The spin lattice relaxation time and the spectral diffusion time of the synthetic melanin are very long (4.3 ms and 101 microseconds, respectively, in the solid state), and the ESR signal saturates readily at low microwave powers. On the other hand, ESR spectra of natural melanins from the tissues chosen for this study, as well as those of synthetic melanins which contain Fe3+ of g = 4.3 and Mn2+ of g = 2, are relatively difficult to saturate compared with samples without such metal ions. These results show clearly that a large part of those two metal ions in sites responsible for the ESR spectral components with these particular g values are coordinated to melanin in melanin-containing tissue, and modify the magnetic relaxation behavior of the melanin. Accumulations of these metal ions in melanins are different from system to system, and they increase in the order: hair (black), retina and choroid (brown), malignant melanoma of eye and skin, and lentigo and nevus of skin.     

Isolation and biophysical studies of natural eumelanins: applications of imaging technologies and ultrafast spectroscopy

The major pigments found in the skin, hair, and eyes of humans and other animals are melanins. Despite significant research efforts, the current understanding of the molecular structure of melanins, the assembly of the pigment within its organelle, and the structural consequences of the association of melanins with protein and metal cations is limited. Likewise, a detailed understanding of the photochemical and photophysical properties of melanins has remained elusive. Many types of melanins have been studied to date, including natural and synthetic model pigments. Such studies are often contradictory and to some extent the diversity of systems studied may have detracted from the development of a basic understanding of the structure and function of the natural pigment. Advances in the understanding of the structure and function of melanins require careful characterization of the pigments examined so as to assure the data obtained may be relevant to the properties of the pigment in vivo. To address this issue, herein the influence of isolation procedures on the resulting structure of the pigment is examined. Sections describing the applications of new technologies to the study of melanins follow this. Advanced imaging technologies such as scanning probe microscopies are providing new insights into the morphology of the pigment assembly. Recent photochemical studies on photoreduction of cytochrome c by different mass fraction of sonicated natural melanins reveal that the photogeneration of reactive oxygen species (ROS) depends upon aggregation of melanin. Specifically, aggregation mitigates ROS photoproduction by UV-excitation, suggesting the integrity of melanosomes in tissue may play an important role in the balance between the photoprotective and photodamaging behaviors attributed to melanins. Ultrafast laser spectroscopy studies of melanins are providing insights into the time scales and mechanisms by which melanin dissipates absorbed light energy.     

Study of photochemical and surface-active melanins of some representatives of black fungi

The absorption spectra of melanins isolated from some black ascomycetes, as well as of synthetic melanin and natural melanin from Sepia officinalis, were recorded in the long-wavelength ultraviolet region A (320 nm < lambda < 400 nm) and in the blue-violet region of the electromagnetic spectrum at illumination intensities varying from 0.02 to 1 mW/cm2. The photochemical properties of fungal melanins were found to be dependent on both the producing strain and the conditions of its cultivation. The fungal melanins are more susceptible to photomodification and more biologically active than the synthetic melanin, indicating that these properties may be related. The data obtained suggest that the fungal melanins susceptible to photomodification possess higher biological activity than commercial melanins.     

Structural differences in unbleached and mildly-bleached synthetic tyrosine-derived melanins identified by scanning probe microscopies

Using scanning tunneling microscopy (STM), we have imaged two types of mildly-bleached, synthetic tyrosine-derived melanins for comparison with the unbleached melanin from which they were prepared. These mildly-bleached melanins were generated by mild oxidation of the unbleached melanin, using either basic hydrogen peroxide or air/light. The unbleached melanin, and two mildly-bleached melanins, were independently deposited from very dilute tetrahydrofuran (THF) solutions onto highly oriented pyrolytic graphite (HOPG) substrate for STM imaging. Lateral dimensions (23 A, average of two directions) of structures from each of the three samples showed no differences. However, structures from both mildly-bleached melanins showed similar dramatic decreases (from approximately 15 A to approximately 5 A) in their STM-measured apparent heights, compared with structures from the unbleached melanin sample. These STM observations are compatible with structural models for unbleached and mildly-bleached melanins, incorporating a three-dimensional structure for unbleached melanin composed of multi-layered, pi-pi-stacked, carboxylic and amino variants of polyaromatic polymeric sheets. The STM-observed decrease in apparent heights after mild oxidation, which we associate with a change in stack height, has been confirmed by experiments using tapping mode atomic force microscopy (TM-AFM) for the unbleached and mildly-hydrogen-peroXide-bleached melanins (from approximately 14 A to approximately 6 A). In these TM-AFM experiments, the melanins were deposited directly onto magnesium cation-treated glass substrates in contact with methanolic solutions of each of the melanins. We interpret our mild-bleaching results as an oxidative conversion of the multi-layered, stacked sheets of mainly carboxylic and amino variants of polyquinhydrone-like moieties, to largely de-stacked, mildly-bleached melanin sheets. These oxidized and, hence, electron-deficient sheets should not readily form multi-layered, pi-pi interacting stacks, but instead appear to be either single-layer polyquinone sheets or, at most, double-layer polyquinhydrone sheets. The effects of such de-stacking on in vivo melanin photoprotection, and structural similarities between melanin derived from natural sources and the synthetic melanin samples used in this work are discussed.     

Interaction of radicals from water radiolysis with melanin

Melanins are considered to be natural photoprotectors in the melanocytes and keratinocytes of the skin. These pigments have also been suggested to play an important role in protection of melanin-containing cells against ionising radiation. Various mechanisms have been proposed to explain the protective role of melanin which invoke the radical scavenging properties of the polymer. In the present work the reactions of melanins with radicals generated in aqueous media by pulse radiolysis have been studied. Time-resolved changes in absorbance of the melanin or the radical species were recorded at selected wavelengths. Experiments were carried out on synthetic dopa- and 5-S-cysteinyldopa-melanins and a natural melanin in phosphate buffer (pH 7.4). Under the conditions employed, melanin reacted predominantly with either oxidising (OH., N3.) or reducing (eaq-, CO2-) species. We were also able to monitor the interaction of melanin with superoxide radical, which was reducing in this case. Detailed analysis of transient changes in melanin absorbance, detected at different wavelengths, was demonstrated to be a convenient method for studying redox processes of this substance, as shown by model experiments using ferricyanide and dithionite as oxidising and reducing agents, respectively. Among the radicals studied, OH. exhibited the strongest reactivity with melanins. Apparent rate constants for the reactions of radicals with autoxidative dopa-melanin (1.5 X 10(9) M-1 X s-1, 2.6 X 10(8) M-1 X s-1, 1.8 X 10(8) M-1 X s-1, 5 X 10(5) M-1 X s-1, 10(6)-10(7) M-1 X s-1 for OH., eaq-, N.3. O2- and CO2-, respectively) are reported. The reactivity of melanins with radicals from water radiolysis and their effect on pigment properties are discussed in terms of the structure and possible biological role of the pigments.     

Free radical scavenging properties of melanin interaction of eu- and pheo-melanin models with reducing and oxidising radicals

The human skin and eye melanin is commonly viewed as an efficient photoprotective agent. To elucidate the molecular mechanism of the melanin-dependent photoprotection, we studied the interaction of two synthetic melanins, dopa-melanin and cysteinyldopa-melanin, with a wide range of oxidising and reducing free radicals using the pulse radiolysis technique. We have found that although both types of free radicals could efficiently interact with the synthetic melanins, their radical scavenging properties depended, in a complex way, on the redox potential, the electric charge and the lifetime of the radicals. Repetitive pulsing experiments, in which the free radicals, probing the polymer redox sites, were generated from four different viologens, indicated that the eumelanin model had more reduced than oxidised groups accessible to reaction with the radicals. Although with many radicals studied, melanin interacted via simple one-electron transfer processes, the reaction of both melanins with the strongly oxidising peroxyl radical from carbon tetrachloride, involved radical addition. Our study suggests that the free radical scavenging properties of melanin may be important in the protection of melanotic cells against free radical damage, particularly if the reactive radicals are generated in close proximity to the pigment granules.     

Natural,semisynthetic and synthetic tyrosinase inhibitors

Tyrosinase plays a pivotal role in the synthesis of melanin pigment synthesis on skin utilizing tyrosine as a substrate. Melanin is responsible for the protection against harmful ultraviolet irradiation, which can cause significant pathological conditions, such as skin cancers. However, it can also create esthetic problems when accumulated as hyperpigmented spots. Various skin-whitening ingredients which inhibit tyrosinase activity have been identified. Some of them, especially ones with natural product origins, possess phenolic moiety and have been employed in cosmetic products. Semi-synthetic and synthetic inhibitors have also been developed under inspiration of the natural inhibitors yet some of which have no phenolic groups. In this review, tyrosinase inhibitors with natural, semi-synthetic and synthetic origins are listed up with their structures, activities and characteristics. Further, a recent report on the adverse effect of a natural melanin synthesis inhibitor which was included in skin-whitening cosmetics is also briefly discussed.     

The Photochemical and Surface-Active Properties of Melanins Isolated from Some Black Fungi

The absorption spectra of melanins isolated from some black ascomycetes, as well as of synthetic melanin and natural melanin from Sepia officinalis, were recorded in the long-wavelength ultraviolet region A (320 nm < < 400 nm) and in the blue–violet region of the electromagnetic spectrum at illumination intensities varying from 0.02 to 1 mW/cm². The photochemical properties of fungal melanins were found to be dependent on both the producing strain and the conditions of its cultivation. The fungal melanins are more susceptible to photomodification and more biologically active than the synthetic melanin, indicating that these properties may be related. The data obtained suggest that the fungal melanins susceptible to photomodification possess higher biological activity than commercial melanins.     

Structural Differences in Unbleached and Mildly‐Bleached Synthetic Tyrosine‐Derived Melanins Identified by Scanning Probe Microscopies

Using scanning tunneling microscopy (STM), we have imaged two types of mildly‐bleached, synthetic tyrosine‐derived melanins for comparison with the unbleached melanin from which they were prepared. These mildly‐bleached melanins were generated by mild oxidation of the unbleached melanin, using either basic hydrogen peroxide or air/light. The unbleached melanin, and two mildly‐bleached melanins, were independently deposited from very dilute tetrahydrofuran (THF) solutions onto highly oriented pyrolytic graphite (HOPG) substrate for STM imaging. Lateral dimensions (23 Å, average of two directions) of structures from each of the three samples showed no differences. However, structures from both mildly‐bleached melanins showed similar dramatic decreases (from ～15 Å to ～5 Å) in their STM‐measured apparent heights, compared with structures from the unbleached melanin sample. These STM observations are compatible with structural models for unbleached and mildly‐bleached melanins, incorporating a three‐dimensional structure for unbleached melanin composed of multi‐layered, Π–Π‐stacked, carboxylic and amino variants of polyaromatic polymeric sheets. The STM‐observed decrease in apparent heights after mild oxidation, which we associate with a change in stack height, has been confirmed by experiments using tapping mode atomic force microscopy (TM‐AFM) for the unbleached and mildly‐hydrogen‐peroxide‐bleached melanins (from ～14 Å to ～6 Å). In these TM‐AFM experiments, the melanins were deposited directly onto magnesium cation‐treated glass substrates in contact with methanolic solutions of each of the melanins. We interpret our mild‐bleaching results as an oxidative conversion of the multi‐layered, stacked sheets of mainly carboxylic and amino variants of polyquinhydrone‐like moieties, to largely de‐stacked, mildly‐bleached melanin sheets. These oxidized and, hence, electron‐deficient sheets should not readily form multi‐layered, Π–Π interacting stacks, but instead appear to be either single‐layer polyquinone sheets or, at most, double‐layer polyquinhydrone sheets. The effects of such de‐stacking on in vivo melanin photoprotection, and structural similarities between melanin derived from natural sources and the synthetic melanin samples used in this work are discussed.     

The effect of melanins on oxidation of lecithin in liposomal membranes

Lecithin peroxidation in liposomal membranes induced by UV light was studied in the presence of natural eye melanin and synthetic melanins prepared from various precursors. It was shown that melanins inhibited lecithin photooxidation, and that the extent of this effect strongly depended on the type and concentration of melanin. Comparative study indicated that melanin obtained from adrenolutin was the most effective antioxidant. The ability to inhibit lipid peroxidation depends both on the concentration of paramagnetic centers in the melanin polymer and the accessibility of these centers for free radicals formed during irradiation of liposomes.     

Physical Studies on Melanins: II. X-Ray Diffraction

A number of purified natural and synthetic melanins have been examined by X-ray diffraction. A consistent finding with all samples was the lack of structure in the diffraction pattern corresponding to any significant crystallinity in these melanin preparations. A diffuse ring, centered at a Bragg spacing of 3.4 A was consistently found in samples of melanin from animal sources, and a similar ring at 4.2 A in all melanins obtained from plants. Models for these two polymer types, based upon the current concept that they primarily involve indole and catechol monomeric units respectively, were then evaluated by a Monte Carlo method. From the comparison of the observed spacings with the calculated ones it was concluded that the 4.2 A spacing in the catechol melanins is probably related to the average interaction between adjacent monomeric units, with mutually random orientations. The 3.4 A spacing observed in indole melanins appears to derive from the tendency of indole monomers (probably of adjacent chains) tending to aggregate in near parallel stacks. Some randomness in the form of translations and rotations parallel to the planar groups is consistent with the diffraction patterns. An interesting finding was that the diffraction pattern of synthetic melanin prepared by the alkaline auto-oxidation of catechol gave the 3.4 A spacing found in the indole melanins of natural origin.     

Melanin accelerates the tyrosinase-catalyzed oxygenation of p-hydroxyanisole (MMEH)

Although pigment melanin has long been though of as "inert," recent work has attested to its chemical reactivity. In this communication, we report that either commercial synthetic melanin prepared by persulfate oxidation of tyrosine ("Sigma melanin") or sepia melanin extracted from cuttlefish markedly accelerates the in vitro oxygenation of p-hydroxyanisole (MMEH), catalyzed by mushroom or B-16 melanoma tyrosinase. Kinetics of 4-methoxy-1,2-benzoquinone formation (lambda max = 413 nm) or of molecular O2 uptake were biphasic, with an initial slow rate ("lag time") followed by a fast linear increase. The biphasic response reflects an initial slow hydroxylation followed by a fast dehydrogenation. Added melanin markedly decreased the lag time but had little effect on subsequent dehydrogenation. Similar effects were observed for tyrosine itself. A complex between MMEH and melanin appears to be the "active" species in these reactions. The results indicate that melanin acts as an electron conduit, which accepts electrons from the substrate and transfers them to tyrosinase. The magnitude of the effect depends on the type of melanin as well as on its oxidation state. Kinetic analysis indicates that both melanins are very efficient at transferring electron to tyrosinase, and that Sigma melanin is roughly threefold more efficient than sepia melanin. The qualitative similarity of reaction between the synthetic and "natural" melanins suggests that the former may serve as a first approximation to the in vivo situation. On the other hand, the observed quantitative differences and the sensitivity of these results to the chemical state of melanin suggests that this methodology might eventually be adapted as a non-destructive probe of melanin in situ.(ABSTRACT TRUNCATED AT 250 WORDS)     

Free radicals from eumelanins: Quantum yields and wavelength dependence

Formation of light-induced free radicals from natural eumelanin (from bovine eyes) and synthetic melanin (from oxidation of 3,4-dihydroxyphenylalanine) has been studied by electron spin resonance spectroscopy. Action spectra measured for natural melanins are very similar to that found for synthetic melanin, and are unaffected by the removal of associated protein. A comparison of action spectra with optical absorbance spectra shows that the former has a more marked wavelength dependence, suggesting that the chromophore that is most active in free-radical production is not the major melanin chromophore that absorbs visible light. Measurements of quantum yields for freeradical production have been made over a wavelength range from 600 to 230 nm. The efficiency of radical production from natural eumelanin is about three times greater than from the synthetic material. Although production of the melanin radicals detected is independent of oxygen, some correlation with oxygen consumption is evident; quantum yields for radical production are approximately three times those for oxygen consumption obtained under similar conditions. Possible reasons for this are discussed.     

Effect of melanins from black yeast fungi on cultured human cells. I. Proliferation of keratinocytes and fibroblasts

Results of screening of the influence exerted by yeast black melanin on the proliferation of human skin keratinocytes and embryonic fibroblasts are presented. The optimal concentration of the investigated melanins was found to be within 0.005 and 0.0001 mg/ml. 17 samples of DHN-melanin from black yeast and 2 commercial samples of [symbol: see text]OPA-melanin (natural and synthetic) were investigated. It was established that keratinocyte proliferation was inhibited by 3 black yeast melanin samples; the influence of other 14 samples was the same as in the control. Keratinocyte proliferation was stimulated only by a commercial sample of natural [symbol: see text]OPA-melanin at concentration 0.005 mg/ml. The synthetic melanin at concentrations 0.005 and 0.001 mg/ml inhibited keratinocyte proliferation. Of the 17 investigated black yeast melanin samples, only one sample stimulated fibroblast proliferation at concentration 0.005 mg/ml. Three other samples inhibited the proliferation; of these one sample did it at all used concentrations, and two samples at concentration 0.0001 mg/ml. The rest 13 samples of black yeast DHN-melanins and the synthetic [symbol: see text]OPA-melanin did not differ in either action from the control.     

Spectroscopic studies of chemically modified synthetic melanins

The infrared and electron spin resonance spectra of synthetic 3,4-dihydroxyphenylalanine (DOPA) and tyrosine melanins and chemically modified melanin samples were determined, and it was shown that unmodified and reduced DOPA melanins exhibited similar ir spectra. Oxidized DOPA melanins showed a higher number of carboxy groups in the sample. A significant increase of free radical content in reduced DOPA melanin and a decrease of free radical content in oxidized DOPA melanin in comparison to unmodified samples were demonstrated by the use of ESR methodology. Methylation of tyrosine melanin with an excess of diazomethane gave very rich ir spectra as compared to melanins methylated with methanol saturated by gaseous HCl. In tyrosine melanin samples the esterification of carboxy groups with methanol caused a decrease in the free radical content. When diazomethane was used, the methylated melanin samples had free radical levels reduced to only about 4% of the total observed for unmodified tyrosine melanin.     

Computational model for pathway reconstruction to unravel the evolutionary significance of melanin synthesis

Melanogenesis is a complex multistep process of high molecular weight melanins production by hydroxylation and polymerization of polyphenols. Melanins have a wide range of applications other than being a sun - protection pigment. Melanogenesis pathway exists from prokaryotes to eukaryotes. It has evolved over years owing to the fact that the melanin pigment has different roles in diverse taxa of organisms. Melanin plays a pivotal role in the existence of certain bacteria and fungi whereas in higher organisms it is a measure of protection against the harmful radiation. We have done a detailed study on various pathways known for melanin synthesis across species. It was divulged that melanin production is not restricted to tyrosine but there are other secondary metabolites that synthesize melanin in lower organisms. Furthermore the phylogenetic study of these paths was done to understand their molecular and cellular development. It has revealed that the melanin synthesis paths have co-evolved in several groups of organisms. In this study, we also introduce a method for the comparative analysis of a metabolic pathway to study its evolution based on similarity between enzymatic reactions.     

Ability of melanins to protect against the radiolysis of thymine and thymidine

Individuals with black skin rarely get skin cancer, and melanomas, tumors arising from pigmented cells, are generally resistant to radiation therapy. The role of melanin in these two phenomena has not been defined, but oxygen-radical species have been implicated in both effects. These studies were undertaken to determine the ability of various melanins to compete for ionizing radiation-produced radicals which destroy nucleic acid bases. The ability of Sigma eumelanin (S-eumelanin) to protect against the radiolysis of thymidine in buffered solutions was compared to the protective ability of seven amino acids, including melanin precursors; bovine serum albumin, as a model protein; ficoll, as a model polysaccharide; and DNA. Both proteins and polysaccharides are known to scavenge hydroxyl radicals in cells. The concentration of thymidine after exposure to gamma radiation was determined by High Performance Liquid Chromatography (HPLC) analysis after removal of insoluble melanin by acid precipitation. S-eumelanin was more effective at competing with thymidine for free radicals than bovine serum albumin, Ficoll, or DNA, but less effective than certain of the small molecules. Several of the above compounds were also examined for ability to protect against thymine radiolysis. In addition, melanins from other sources were compared to S-eumelanin. Of these, enzymatically synthesized phaeomelanin was the most effective. The results indicate that melanins can compete for base- and nucleoside-damaging free radicals more effectively than other cellular macromolecules. Of the small molecules, the phenolic compounds had the greatest scavenging ability. In vivo, melanins are found in melanosomes bound to protein. Therefore, the relevance of these findings to the photo- and radiobiology of melanins in vivo has yet to be determined.     

Reexamination of the structure of eumelanin

The generally accepted concept that the black melanin eumelanin is made mostly from 5,6-dihydroxyindole but not from 5,6-dihydroxyindole-2-carboxylic acid (DHIC) was reexamined by comparison of synthetic and natural eumelanins. The analytical methods used were elemental analysis and determination of the carboxyl group by acid treatment to yield CO2 and by permanganate oxidation to yield pyrrole-2,3,5-tricarboxylic acid. It was found that DHIC-derived monomer units comprise only approx. 10% of enzymically prepared dopa-melanins but as much as a half of intact, natural eumelanins. The results also show that dopa-melanins prepared at higher pH retain higher percentages of the carboxyl group of dopa and contain higher percentages of pyrrole units, and that melanins are decomposed to a significant extent on acid treatment, the method commonly used to isolate melanins from natural sources.     

Analysis of the structure of synthetic and natural melanins by solid-phase NMR

The structures of one synthetic and two natural melanins are examined by solid-state NMR using cross polarization, magic angle sample spinning, and high-power proton decoupling. The structural features of synthetic dopa melanin are compared to those of melanin from malignant melanoma cells grown in culture and sepia melanin from squid ink. Natural abundance 13C and 15N spectra show resonances consistent with known pyrrolic and indolic structures within the heterogeneous biopolymer; 13C spectra indicate the presence of aliphatic residues in all three materials. These solid-phase experiments illustrate the promise of solid-phase NMR for elucidating structural information from insoluble biomaterials.