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1.  Dispersive Raman spectroscopy allows the identification and quantification of melanin types 
Ecology and Evolution  2015;5(7):1425-1431.
Melanins are the most prevalent pigments in animals and are involved in visual communication by producing colored traits that often evolve as intraspecific signals of quality. Identifying and quantifying melanins are therefore essential to understand the function and evolution of melanin-based signals. However, the analysis of melanins is difficult due to their insolubility and the lack of simple methods that allow the identification of their chemical forms. We recently proposed the use of Raman spectroscopy as a simple, noninvasive technique that can be used to identify and quantify melanins in feathers and hairs. Contrarily, other authors later stated that melanins are characterized by a lack of defined Raman signals. Here, we use confocal Raman microscopy to confirm previous analyses showing that the two main chemical forms of melanins (eumelanin and pheomelanin) exhibit distinct Raman signal and compare different excitation wavelengths to analyze synthetic pheomelanin and natural melanins in feathers of different species of birds. Our analyses indicate that only laser excitation wavelengths below 1064 nm are useful for the analysis of melanins by Raman spectroscopy, and only 780-nm laser in the case of melanins in feathers. These findings show that the capacity of Raman spectroscopy to distinguish different chemical forms of melanins depends on laser power and integration time. As a consequence, Raman spectroscopy should be applied after preliminar analyses using a range of these parameters, especially in fragile biological tissues such as feathers.
doi:10.1002/ece3.1453
PMCID: PMC4395172  PMID: 25897382
Eumelanin; feathers; pheomelanin; Raman spectroscopy
2.  Near-Infrared Excited State Dynamics of Melanins: The Effects of Iron Content, Photo-Damage, Chemical Oxidation, and Aggregate Size 
The Journal of Physical Chemistry. a  2014;118(6):993-1003.
Ultrafast pump–probe measurements can discriminate the two forms of melanin found in biological tissue (eumelanin and pheomelanin), which may be useful for diagnosing and grading melanoma. However, recent work has shown that bound iron content changes eumelanin’s pump–probe response, making it more similar to that of pheomelanin. Here we record the pump–probe response of these melanins at a wider range of wavelengths than previous work and show that with shorter pump wavelengths the response crosses over from being dominated by ground-state bleaching to being dominated by excited-state absorption. The crossover wavelength is different for each type of melanin. In our analysis, we found that the mechanism by which iron modifies eumelanin’s pump–probe response cannot be attributed to Raman resonances or differences in melanin aggregation and is more likely caused by iron acting to broaden the unit spectra of individual chromophores in the heterogeneous melanin aggregate. We analyze the dependence on optical intensity, finding that iron-loaded eumelanin undergoes irreversible changes to the pump–probe response after intense laser exposure. Simultaneously acquired fluorescence data suggest that the previously reported “activation” of eumelanin fluorescence may be caused in part by the dissociation of metal ions or the selective degradation of iron-containing melanin.
doi:10.1021/jp4107475
PMCID: PMC3983346  PMID: 24446774
3.  Near-Infrared Excited State Dynamics of Melanins: The Effects of Iron Content, Photo-Damage, Chemical Oxidation, and Aggregate Size 
The journal of physical chemistry. A  2014;118(6):993-1003.
Ultrafast pump–probe measurements can discriminate the two forms of melanin found in biological tissue (eumelanin and pheomelanin), which may be useful for diagnosing and grading melanoma. However, recent work has shown that bound iron content changes eumelanin’s pump–probe response, making it more similar to that of pheomelanin. Here we record the pump–probe response of these melanins at a wider range of wavelengths than previous work and show that with shorter pump wavelengths the response crosses over from being dominated by ground-state bleaching to being dominated by excited-state absorption. The crossover wavelength is different for each type of melanin. In our analysis, we found that the mechanism by which iron modifies eumelanin’s pump–probe response cannot be attributed to Raman resonances or differences in melanin aggregation and is more likely caused by iron acting to broaden the unit spectra of individual chromophores in the heterogeneous melanin aggregate. We analyze the dependence on optical intensity, finding that iron-loaded eumelanin undergoes irreversible changes to the pump–probe response after intense laser exposure. Simultaneously acquired fluorescence data suggest that the previously reported “activation” of eumelanin fluorescence may be caused in part by the dissociation of metal ions or the selective deeradation of iron-containine melanin.
doi:10.1021/jp4107475
PMCID: PMC3983346  PMID: 24446774
4.  Chemosorption of radiometals of interest to nuclear medicine by synthetic melanins 
Nuclear medicine and biology  2008;35(3):353-357.
Introduction
Melanins are high molecular weight pigments that are ubiquitous in nature and can also be synthesized in the laboratory from the variety of precursors. Melanins possess numerous interesting physico-chemical characteristics including electromagnetic radiation absorption properties and the ability to chelate metals. We have recently reported that melanin has remarkable ionizing radiation shielding properties, possibly because it can interact with photons via Compton scattering. We hypothesized that, if administered internally, in addition to radiation shielding, melanin could play a beneficial role by scavenging various radionuclides.
Methods
Three melanins were synthesized from dopamine, 3,4-dihydroxyphenylalanine (L-Dopa) and from combination of L-Cysteine and L-Dopa. For control synthetic melanin made from tyrosine polymerization (Sigma) was used. Melanins were characterized by elemental analysis. The chemosorption of 111In, 225Ac and 213Bi by melanins was studied at 37°C for up to 48 hrs.
Results
The C to N molar ratios for dopamine, L-Dopa and tyrosine melanins were very close at 7.92, 8.39, and 8.48, respectively, while in mixed L-cysteine/L-Dopa melanin that ratio was much lower at 3.63. This mixed melanin also contained 22.33% sulfur, thus confirming incorporation of S-containing motifs into its structure. Dopamine, L-Dopa and tyrosine melanins were very similar in their ability to decrease the activity of 111In, 225Ac and 213Bi and their radioactive daughters in the supernatants more than 10-fold in comparison with the starting levels while mixed L-cysteine/L-Dopa melanin was able to chemosorb only 111In.
Conclusions
We have demonstrated that synthetic melanins made of diverse precursors can chemosorb 111In, 213Bi and 225Ac with dopamine, L-dopa and tyrosine melanins being the most efficient towards all three of these radionuclides. Such properties of synthetic melanins can contribute to the development of the novel melanin-based radioprotective materials.
doi:10.1016/j.nucmedbio.2007.12.006
PMCID: PMC2516407  PMID: 18355691
melanin; chemosorption; 225-Actinium; 213-Bismuth; 111-Indium; radiological attack
5.  Spiny Mice Modulate Eumelanin to Pheomelanin Ratio to Achieve Cryptic Coloration in “Evolution Canyon,” Israel 
PLoS ONE  2010;5(1):e8708.
Background
Coat coloration in mammals is an explicit adaptation through natural selection. Camouflaging with the environment is the foremost evolutionary drive in explaining overall coloration. Decades of enquiries on this topic have been limited to repetitive coat color measurements to correlate the morphs with background/habitat blending. This led to an overwhelming endorsement of concealing coloration as a local phenotypic adaptation in animals, primarily rodents to evade predators. However, most such studies overlooked how rodents actually achieve such cryptic coloration. Cryptic coloration could be attained only through optimization between the yellow- to brown-colored “pheomelanin” and gray to black-colored “eumelanin” in the hairs. However, no study has explored this conjecture yet. “Evolution Canyon” (EC) in Israel is a natural microscale laboratory where the relationship between organism and environment can be explored. EC is comprised of an “African” slope (AS), which exhibits a yellow-brownish background habitat, and a “European” slope (ES), exhibiting a dark grayish habitat; both slopes harbor spiny mice (Acomys cahirinus). Here, we examine how hair melanin content of spiny mice living in the opposing slopes of EC evolves toward blending with their respective background habitat.
Methodology/Principal Findings
We measured hair-melanin (both eumelanin and pheomelanin) contents of 30 spiny mice from the EC using high-performance liquid chromatography (HPLC) that detects specific degradation products of eumelanin and pheomelanin. The melanin pattern of A. cahirinus approximates the background color of the slope on which they dwell. Pheomelanin is slightly (insignificantly) higher in individuals found on the AS to match the brownish background, whereas individuals of the ES had significantly greater eumelanin content to mimic the dark grayish background. This is further substantiated by a significantly higher eumelanin and pheomelanin ratio on the ES than on the AS.
Conclusion/Significance
It appears that rodents adaptively modulate eumelanin and pheomelanin contents to achieve cryptic coloration in contrasting habitats even at a microscale.
doi:10.1371/journal.pone.0008708
PMCID: PMC2806840  PMID: 20090935
6.  Effect of Eye Pigmentation on Transscleral Drug Delivery 
Purpose
To determine the influence of eye pigmentation on transscleral retinal delivery of celecoxib.
Methods
Melanin content in ocular tissues of both the strains was determined by sodium hydroxide solubilization method. The affinity of celecoxib to synthetic and natural melanin was estimated by co-incubating celecoxib and melanin in isotonic phosphate-buffered saline. The binding affinity (k) and the maximum binding (rmax) for celecoxib to both natural and synthetic melanin were estimated. Suspension of celecoxib (3 mg/rat) was injected periocularly into one eye of Sprague-Dawley (SD, albino) and Brown Norway (BN, pigmented) rats. The animals were euthanatized at the end of 0.25, 0.5, 1, 2, 3, 4, 8, or 12 hours after the drug was administered, and celecoxib levels in ocular tissues (sclera, choroid-RPE, retina, vitreous, lens, and cornea) were estimated with an HPLC assay. In addition, celecoxib-poly(lactide) microparticles (750 μg drug/rat) were administered periocularly in SD and BN rats, and celecoxib levels in these eye tissues were assessed on day 8, to determine the effectiveness of the sustained release system.
Results
The rmax and k for celecoxib’s binding to natural melanin were (3.92 ± 0.06) × 10−7 moles/mg of melanin and (0.08 ± 0.01) × 106 M−1, respectively. The affinity and the extent of celecoxib’s binding to natural melanin were not significantly different from those observed with synthetic melanin. The concentrations of melanin in choroid-RPE, sclera, and retina of BN rats were 200 ± 30, 12 ± 4, and 3 ± 0.2 μg/mg tissue, respectively. Melanin was not detectable in the vitreous, lens, and cornea of BN rats. In SD rats, melanin was not detected in all tissues assessed except in the choroid-RPE, wherein melanin-like activity was 100-fold less than in BN rats. The area under the curve (AUC) for tissue concentration versus time profiles for animals administered with celecoxib suspension was not significantly different between the two strains for sclera, cornea, and lens. However, the retinal (P = 0.001) and vitreal (P = 0.001) AUCs of celecoxib in the treated eyes were approximately 1.5-fold higher in SD rats than in BN rats. Further, the choroid-RPE AUC in the treated and untreated eyes, respectively, were 1.5-fold (P = 0.001) and 2-fold (P = 0.0001) higher in BN rats than in SD rats. With celecoxib-poly(lactide) microparticles, choroid-RPE, retina, and vitreous concentrations on day 8 exhibited similar trends in differences between the two strains, with the differences being greater than those recorded for the celecoxib suspension.
Conclusions
Transscleral retinal and vitreal drug delivery of lipophilic celecoxib is significantly lower in pigmented rats than in albino rats. This difference may be attributable to significant binding of celecoxib to melanin and its accumulation/retention in the melanin-rich choroid-RPE of pigmented rats. The hindrance of retinal and vitreal drug delivery by the choroid-RPE in pigmented rats is also true of sustained-release microparticle systems.
doi:10.1167/iovs.07-0214
PMCID: PMC3324932  PMID: 18172110
7.  Protection of Melanized Cryptococcus neoformans from Lethal Dose Gamma Irradiation Involves Changes in Melanin's Chemical Structure and Paramagnetism 
PLoS ONE  2011;6(9):e25092.
Certain fungi thrive in highly radioactive environments including the defunct Chernobyl nuclear reactor. Cryptococcus neoformans (C. neoformans), which uses L-3,4-dihydroxyphenylalanine (L-DOPA) to produce melanin, was used here to investigate how gamma radiation under aqueous aerobic conditions affects the properties of melanin, with the aim of gaining insight into its radioprotective role. Exposure of melanized fungal cell in aqueous suspensions to doses of γ-radiation capable of killing 50 to 80% of the cells did not lead to a detectable loss of melanin integrity according to EPR spectra of melanin radicals. Moreover, upon UV-visible (Xe-lamp) illumination of melanized cells, the increase in radical population was unchanged after γ-irradiation. Gamma-irradiation of frozen cell suspensions and storage of samples for several days at 77 K however, produced melanin modification noted by a reduced radical population and reduced photoresponse. More direct evidence for structural modification of melanin came from the detection of soluble products with absorbance maxima near 260 nm in supernatants collected after γ-irradiation of cells and cell-free melanin. These products, which include thiobarbituric acid (TBA)-reactive aldehydes, were also generated by Fenton reagent treatment of cells and cell-free melanin. In an assay of melanin integrity based on the metal (Bi+3) binding capacity of cells, no detectable loss in binding was detected after γ-irradiation. Our results show that melanin in C. neoformans cells is susceptible to some damage by hydroxyl radical formed in lethal radioactive aqueous environments and serves a protective role in melanized fungi that involves sacrificial breakdown.
doi:10.1371/journal.pone.0025092
PMCID: PMC3178601  PMID: 21966422
8.  Effect of Metal Ions on Melanin – Local Anaesthetic Drug Complexes 
The affinity of melanin biopolymers for metal ions, drugs and other organic compounds is an important factor in the etiology of toxic retinopathy, hiperpigmentation, otic lesions and irreversible extrapyramidal disorders. The aim of the presented work was to examine the interaction of local anaesthetic drugs used in ophthalmology with model DOPA-melanin in the presence of metal ions. It has been demonstrated that the analyzed drugs form complexes with melanin biopolymer. Based on the .values of association constants,, the following order of drugs affinity to melanin was found: tetracaine > procaine >> bupivacaine > lidocaine. It has also been shown that Cu2+ and Zn2+ ions administered to DOPA-melanin before complexing with drugs decrease the total amount of local anaesthetics bound to melanin. The blocking of some active centers in melanin molecules by metal ions, which potentially exist in living systems, may change the clinical therapeutic efficiency of the analyzed local anaesthetic drugs.
doi:10.1155/S1565363303000098
PMCID: PMC2267052  PMID: 18365047
9.  HGF/SF Increases Number of Skin Melanocytes but Does Not Alter Quality or Quantity of Follicular Melanogenesis 
PLoS ONE  2013;8(11):e74883.
Melanins are an important factor determining the vulnerability of mammalian skin to UV radiation and thus to UV-induced skin cancers. Transgenic mice overexpressing hepatocyte growth factor/scatter factor (HGF/SF) have extra-follicular dermal melanocytes, notably in the papillary upper dermis, and are susceptible to UV-induced melanoma. Pigmented HGF/SF neonatal mice are more susceptible than albino HGF/SF animals to UVA -induced melanoma, indicating an involvement of melanin in melanoma formation. This raises the question of the effect of transgenic HGF/SF on melanization. We developed a methodology to accurately quantitate both the production of melanin and the efficiency of melanogenesis in normal, and HGF/SF transgenic mice in vivo. Skin and hair shafts of 5 day old and adult (3 week old) C57BL/6-HGF/SF and corresponding C57BL/6 wild type mice were investigated by electron paramagnetic resonance spectroscopy (EPR) to quantitate melanin, by transmission electron microscopy (TEM) for the presence of melanosomes, and by standard histology and by Western blotting and zymography to determine the expression and activity of melanogenesis-related proteins. Eumelanin but no phaeomelanin was detected in transgenic C57BL/6-HGF and C57BL/6 wild type mice. Transgenic HGF/SF overexpression did not change the type of melanin produced in the skin or hair, did not affect the terminal content of melanin production in standard samples of hair and did not influence hair cycle/morphogenesis-related changes in skin thickness. No melanocytes were found in the epidermis and no melanosomes were found in epidermal keratinocytes. HGF/SF transgenic mice thus lack the epidermal melanin UV-protection found in constitutively dark human skin. We conclude that melanocytes in the HGF/SF transgenic mouse, particularly in the papillary dermis, are vulnerable to UVA which interacts with eumelanin but not phaeomelanin to induce melanoma.
doi:10.1371/journal.pone.0074883
PMCID: PMC3819350  PMID: 24223113
10.  1,8-Dihydroxynaphthalene (DHN)-Melanin Biosynthesis Inhibitors Increase Erythritol Production in Torula corallina, and DHN-Melanin Inhibits Erythrose Reductase 
The yeast Torula corallina is a strong erythritol producer that is used in the industrial production of erythritol. However, melanin accumulation during culture represents a serious problem for the purification of erythritol from the fermentation broth. Melanin biosynthesis inhibitors such as 3,4-dihydroxyphenylalanine and 1,8-dihydroxynaphthalene (DHN)-melanin inhibitors were added to the T. corallina cultures. Only the DHN-melanin inhibitors showed an effect on melanin production, which suggests that the melanin formed during the culturing of T. corallina is derived from DHN. This finding was confirmed by the detection of a shunt product of the pentaketide pathway, flaviolin, and elemental analysis. Among the DHN-melanin inhibitors, tricyclazole was the most effective. Supplementation with tricyclazole enhanced the production of erythritol while significantly inhibiting the production of DHN-melanin and DHN-melanin biosynthetic enzymes, such as trihydroxynaphthalene reductase. The erythrose reductase from T. corallina was purified to homogeneity by ion-exchange and affinity chromatography. Purified erythrose reductase was significantly inhibited in vitro in a noncompetitive manner by elevated levels of DHN-melanin. In contrast, the level of erythrose reductase activity was unaffected by increasing concentrations of tricyclazole. These results suggest that supplemental tricyclazole reduces the production of DHN-melanin, which may lead to a reduction in the inhibition of erythrose reductase and a higher yield of erythritol. This is the first report to demonstrate that melanin biosynthesis inhibitors increase the production of a sugar alcohol in T. corallina.
doi:10.1128/AEM.69.6.3427-3434.2003
PMCID: PMC161539  PMID: 12788746
11.  L-DOPA Is an Endogenous Ligand for OA1 
PLoS Biology  2008;6(9):e236.
Albinism is a genetic defect characterized by a loss of pigmentation. The neurosensory retina, which is not pigmented, exhibits pathologic changes secondary to the loss of pigmentation in the retina pigment epithelium (RPE). How the loss of pigmentation in the RPE causes developmental defects in the adjacent neurosensory retina has not been determined, but offers a unique opportunity to investigate the interactions between these two important tissues. One of the genes that causes albinism encodes for an orphan GPCR (OA1) expressed only in pigmented cells, including the RPE. We investigated the function and signaling of OA1 in RPE and transfected cell lines. Our results indicate that OA1 is a selective L-DOPA receptor, with no measurable second messenger activity from two closely related compounds, tyrosine and dopamine. Radiolabeled ligand binding confirmed that OA1 exhibited a single, saturable binding site for L-DOPA. Dopamine competed with L-DOPA for the single OA1 binding site, suggesting it could function as an OA1 antagonist. OA1 response to L-DOPA was defined by several common measures of G-protein coupled receptor (GPCR) activation, including influx of intracellular calcium and recruitment of β-arrestin. Further, inhibition of tyrosinase, the enzyme that makes L-DOPA, resulted in decreased PEDF secretion by RPE. Further, stimulation of OA1 in RPE with L-DOPA resulted in increased PEDF secretion. Taken together, our results illustrate an autocrine loop between OA1 and tyrosinase linked through L-DOPA, and this loop includes the secretion of at least one very potent retinal neurotrophic factor. OA1 is a selective L-DOPA receptor whose downstream effects govern spatial patterning of the developing retina. Our results suggest that the retinal consequences of albinism caused by changes in melanin synthetic machinery may be treated by L-DOPA supplementation.
Author Summary
Albinism is the loss of pigmentation caused by mutations in one of several different genes that alter pigment synthesis by different mechanisms. In the eye, albinism impairs sensory retina development and causes significant vision problems. Regardless of the genetic mutation that causes albinism, the associated vision problems are the same. Interestingly, none of the pigmentation genes are expressed by the sensory retinal cells affected by albinism but by neighboring, retinal pigment epithelial cells (RPE). Furthermore, loss of pigmentation in RPE somehow leads to imprecise retinal development. To investigate this cellular relationship, we studied OA1, which is encoded by a gene in which mutations cause ocular albinism. OA1 is unique among proteins involved with albinism because OA1 is a potential receptor that could participate in signal transduction rather than being a direct member of the pigment synthesis machinery. We show that the ligand for OA1 is L-DOPA, thus removing OA1 from orphan G-protein coupled receptor (GPCR) status. L-DOPA is a by-product of pigment synthesis, indicating that pigment synthesis and OA1 signaling are intertwined. OA1 signaling is highly selective for L-DOPA, and we show that two closely related molecules, dopamine and tyrosine, bind to OA1 but fail to stimulate signaling. We also show that OA1 signaling controls secretion of a potent neuron survival factor. Taken together, our data suggest that all forms of albinism produce the same retinal defects because of a final common pathway through OA1 signaling with downstream effects on RPE neurotrophic factor secretion.
Albinism produces retinal defects, and OA1 is an orphan G-protein-coupled receptor that leads to albinism without acting directly on melanin synthesis. Here the ligand is identified and a mechanism is proposed by which the various forms of albinism signal through OA1, resulting in the same retinal phenotype.
doi:10.1371/journal.pbio.0060236
PMCID: PMC2553842  PMID: 18828673
12.  Invited Review MC1R, Eumelanin and Pheomelanin: their role in determining the susceptibility to skin cancer† 
Photochemistry and photobiology  2014;91(1):188-200.
Skin pigmentation is due to the accumulation of two types of melanin granules in the keratinocytes. Besides being the most potent blocker of ultraviolet radiation (UVR), the role of melanin in photo-protection is complex. This is because one type of melanin called eumelanin is UV absorbent whereas the other, pheomelanin, is photo-unstable and may even promote carcinogenesis. Skin hyperpigmentation may be caused by stress or exposure to sunlight, which stimulates the release of α-melanocyte stimulating hormone (α-MSH) from damaged keratinocytes. Melanocortin 1 receptor (MC1R) is a key signaling molecule on melanocytes that responds to α-MSH by inducing expression of enzymes responsible for eumelanin synthesis. Persons with red hair have mutations in the MC1R causing its inactivation; this leads to a paucity of eumelanin production and makes red-heads more susceptible to skin cancer. Apart from its effects on melanin production, the α-MSH/MC1R signaling is also a potent anti-inflammatory pathway and has been shown to promote anti-melanoma immunity. This review will focus on the role of MC1R in terms of its regulation of melanogenesis and influence on the immune system with respect to skin cancer susceptibility.
doi:10.1111/php.12335
PMCID: PMC4299862  PMID: 25155575
13.  Studies on Synthetic and Natural Melanin and Its Affinity for Fe(III) Ion 
In this work, we measured the metal-binding sites of natural and synthetic dihydroxyindole (DHI) melanins and their respective interactions with Fe(III) ions. Besides the two acid groups detected for the DHI system: catechol (Cat) and quinone-imine (QI), acetate groups were detected in the natural oligomer by potentiometric titrations. At acidic pH values, Fe(III) complexation with synthetic melanin was detected in an Fe(OH)(CatH2Cat) interaction. With an increase of pH, three new interactions occurred: dihydroxide diprotonated catechol, Fe(OH)2(CatH2Cat)−, dihydroxide monoprotonated catechol, [Fe(OH)2(CatHCat)]2−, and an interaction resulting from the association of one quinone-imine and a catechol group, [Fe(OH)2(Qi−)(CatHCat)]3−. In the natural melanin system, we detected the same interactions involving catechol and quinone-imine groups but also the metal interacts with acetate group at pH values lower than 4.0. Furthermore, interactions in the synthetic system were also characterized by infrared spectroscopy by using the characteristic vibrations of catechol and quinone-imine groups. Finally, scanning electronic microscopy (SEM) and energy-dispersive X-ray (EDS) analysis were used to examine the differences in morphology of these two systems in the absence and presence of Fe(III) ions. The mole ratio of metal and donor atoms was obtained by the EDS analysis.
doi:10.1155/2012/712840
PMCID: PMC3521465  PMID: 23251127
14.  The expression of melanin-based plumage is separately modulated by exogenous oxidative stress and a melanocortin 
Melanin-based traits involved in animal communication have been traditionally viewed as occurring under strict genetic control. However, it is generally accepted that both genetic and environmental factors influence melanin production. Medical studies suggest that, among environmental factors influencing melanization, oxidative stress could play a relevant role. On the other hand, genetic control would be exerted by the melanocortin system, and particularly by the alpha-melanocyte-stimulating hormone (α-MSH), which triggers the production of eumelanins (black pigments). To determine how the melanocortin system and an exogenous source of oxidative stress interact in the expression of melanin-based plumage, developing red-legged partridges (Alectoris rufa) were manipulated. Some partridges were injected with α-MSH, while other birds received a pro-oxidant molecule (diquat) in drinking water. Controls and birds receiving both treatments were also studied. Both α-MSH- and diquat-treated individuals presented larger eumelanin-based traits than controls, but α-MSH+diquat-treated birds showed the largest traits, suggesting that oxidative stress and melanocortins promote additive but independent effects. Diquat also induced a decline in the level of a key intracellular antioxidant (glutathione), which is associated with high expression of eumelanin-based signals in other bird species. Some scenarios for the evolution of melanin-based traits in relation to oxidative stress are proposed.
doi:10.1098/rspb.2009.0774
PMCID: PMC2817136  PMID: 19520801
melanocortin system; eumelanin; glutathione; oxidative stress; pheomelanin
15.  Melanins Protect Sporothrix brasiliensis and Sporothrix schenckii from the Antifungal Effects of Terbinafine 
PLoS ONE  2016;11(3):e0152796.
Terbinafine is a recommended therapeutic alternative for patients with sporotrichosis who cannot use itraconazole due to drug interactions or side effects. Melanins are involved in resistance to antifungal drugs and Sporothrix species produce three different types of melanin. Therefore, in this study we evaluated whether Sporothrix melanins impact the efficacy of antifungal drugs. Minimal inhibitory concentrations (MIC) and minimal fungicidal concentrations (MFC) of two Sporothrix brasiliensis and four Sporothrix schenckii strains grown in the presence of the melanin precursors L-DOPA and L-tyrosine were similar to the MIC determined by the CLSI standard protocol for S. schenckii susceptibility to amphotericin B, ketoconazole, itraconazole or terbinafine. When MICs were determined in the presence of inhibitors to three pathways of melanin synthesis, we observed, in four strains, an increase in terbinafine susceptibility in the presence of tricyclazole, a DHN-melanin inhibitor. In addition, one S. schenckii strain grown in the presence of L-DOPA had a higher MFC value when compared to the control. Growth curves in presence of 2×MIC concentrations of terbinafine showed that pyomelanin and, to a lesser extent, eumelanin were able to protect the fungi against the fungicidal effect of this antifungal drug. Our results suggest that melanin protects the major pathogenic species of the Sporothrix complex from the effects of terbinafine and that the development of new antifungal drugs targeting melanin synthesis may improve sporotrichosis therapies.
doi:10.1371/journal.pone.0152796
PMCID: PMC4816517  PMID: 27031728
16.  Temperature-sensitive tyrosinase associated with peripheral pigmentation in oculocutaneous albinism. 
Journal of Clinical Investigation  1991;87(3):1046-1053.
Several types of autosomal recessive oculocutaneous albinism (OCA) are associated with abnormal tyrosinase function and a generalized reduction in or absence of cutaneous and eye melanin. Each is thought to result from a different mutant allele at the tyrosinase locus, with the mutation producing an enzyme with little or no activity in all involved tissues. In this paper, we report a new type of OCA that results from a tyrosinase allele producing a temperature-sensitive enzyme. The proband had white hair in the warmer areas (scalp and axilla) and progressively darker hair in the cooler areas (extremities) of her body. Melanocyte and melanosome architecture were normal. Quantitative hairbulb tyrosinase (dopa oxidase) assay demonstrated a loss of activity above 35-37 degrees C. Plasma pheomelanin and urine eumelanin intermediates were reduced and correlated with hair melanin content. This is the first temperature-sensitive tyrosinase mutation to be reported in humans and is analogous to the Siamese mutation in the cat and the Himalayan mutation in the mouse.
Images
PMCID: PMC329899  PMID: 1900307
17.  “Fifty Shades” of Black and Red or How Carboxyl Groups Fine Tune Eumelanin and Pheomelanin Properties 
Recent advances in the chemistry of melanins have begun to disclose a number of important structure-property-function relationships of crucial relevance to the biological role of human pigments, including skin (photo) protection and UV-susceptibility. Even slight variations in the monomer composition of black eumelanins and red pheomelanins have been shown to determine significant differences in light absorption, antioxidant, paramagnetic and redox behavior, particle morphology, surface properties, metal chelation and resistance to photo-oxidative wear-and-tear. These variations are primarily governed by the extent of decarboxylation at critical branching points of the eumelanin and pheomelanin pathways, namely the rearrangement of dopachrome to 5,6-dihydroxyindole (DHI) and 5,6-dihydroxyindole-2-carboxylic acid (DHICA), and the rearrangement of 5-S-cysteinyldopa o-quinoneimine to 1,4-benzothiazine (BTZ) and its 3-carboxylic acid (BTZCA). In eumelanins, the DHICA-to-DHI ratio markedly affects the overall antioxidant and paramagnetic properties of the resulting pigments. In particular, a higher content in DHICA decreases visible light absorption and paramagnetic response relative to DHI-based melanins, but markedly enhances antioxidant properties. In pheomelanins, likewise, BTZCA-related units, prevalently formed in the presence of zinc ions, appear to confer pronounced visible and ultraviolet A (UVA) absorption features, accounting for light-dependent reactive oxygen species (ROS) production, whereas non-carboxylated benzothiazine intermediates seem to be more effective in inducing ROS production by redox cycling mechanisms in the dark. The possible biological and functional significance of carboxyl retention in the eumelanin and pheomelanin pathways is discussed.
doi:10.3390/ijms17050746
PMCID: PMC4881568  PMID: 27196900
eumelanin; pheomelanin; melanins; 5,6-dihydroxyindoles; 5-S-cysteinyldopa; benzothiazines; dopachrome; melanocortin-1-receptor; antioxidant; pro-oxidant
18.  Regulation of eumelanin / pheomelanin synthesis and visible pigmentation in melanocytes by ligands of the melanocortin 1 receptor 
Pigment cell & melanoma research  2008;21(4):477-486.
The production of melanin in the hair and skin is tightly regulated by the melanocortin 1 receptor (MC1R) whose activation is controlled by 2 secreted ligands, α-melanocyte stimulating hormone (αMSH) and agouti signal protein (ASP). Since melanin is extremely stable, lasting years in biological tissues, the mechanism underlying the relatively rapid decrease in visible pigmentation elicited by ASP is of obvious interest. In this study, the effects of ASP and αMSH on the regulation of melanin synthesis and on visible pigmentation were assessed in normal murine melanocytes and were compared with the quick depigmenting effect of the tyrosinase inhibitor, phenylthiourea (PTU). αMSH increased pheomelanin levels prior to increasing eumelanin content over 4 days of treatment. Conversely, ASP switched off the pigment synthesis pathway, reducing eu- and pheo- melanin synthesis within 1 day of treatment that was proportional to the decrease in tyrosinase protein level and activity. These results demonstrate that the visible depigmentation of melanocytes induced by ASP does not require the degradation of existing melanin but rather is due to the dilution of existing melanin by melanocyte turnover, which emphasizes the importance of pigment distribution to visible color.
doi:10.1111/j.1755-148X.2008.00479.x
PMCID: PMC2562678  PMID: 18627531
eumelanin; pheomelanin; MC1R; ASP; αMSH; PTU; tyrosinase
19.  EPR Studies of DOPA–Melanin Complexes with Netilmicin and Cu(II) at Temperatures in the Range of 105–300 K 
Applied Magnetic Resonance  2012;43(3):341-351.
The application of electron paramagnetic resonance (EPR) spectroscopy in pharmacy of melanin complexes with netilmicin and Cu(II) was presented. The continuous microwave saturation of EPR spectra of DOPA–melanin and the complexes was performed. EPR spectra were measured on an X-band (9.3 GHz) spectrometer at temperatures in the range of 105–300 K. Paramagnetic copper ions decrease the intensity of the EPR lines of melanin’s free radicals. It was found that fast spin–lattice relaxation characterizes DOPA–melanin–Cu(II) complexes. Slow spin–lattice relaxation processes exist in melanin’s paramagnetic centers of DOPA–melanin and DOPA–melanin–netilmicin, [DOPA–melanin–netilmicin]–Cu(II), [DOPA–melanin–Cu(II)]–netilmicin complexes. Spin–lattice relaxation processes are faster at higher temperatures. The homogeneous broadening of EPR lines for melanin complexes was observed. The practical consequences of differences between paramagnetic properties of melanin complexes with netilmicin and the complexes with Cu(II) were discussed.
doi:10.1007/s00723-012-0340-y
PMCID: PMC3459086  PMID: 23144536
20.  Probing Near Infrared Photo-Relaxation Pathways in Eumelanins and Pheomelanins 
The Journal of Physical Chemistry. a  2010;114(43):11483-11491.
Ultraviolet-visible spectroscopy readily discerns the two types of melanin pigments (eumelanin and pheomelanin) although fundamental details regarding the optical properties and pigment heterogeneity are more difficult to disentangle via analysis of the broad featureless absorption spectrum alone. We employed nonlinear transient absorption spectroscopy to study different melanin pigments at near infrared wavelengths. Excited state absorption, ground state depletion and stimulated emission signal contributions were distinguished for natural and synthetic eumelanins and pheomelanins. A starker contrast amongst the pigments is observed in the nonlinear excitation regime because they all exhibit distinct transient absorptive amplitudes, phase shifts and nonexponential population dynamics spanning the femtosecond – nanosecond range. In this manner, different pigments within the pheomelanin subclass were distinguished in synthetic and human hair samples. These results highlight the potential of nonlinear spectroscopies to deliver an in situ analysis of natural melanins in tissue that are otherwise difficult to extract and purify.
doi:10.1021/jp103608d
PMCID: PMC3334281  PMID: 20882951
Melanin; Excited State Dynamics; Human Hair
21.  A melanin-independent interaction between Mc1r and Met signalling pathways is required for HGF-dependent melanoma 
Melanocortin 1 receptor (MC1R) signaling stimulates black eumelanin production through a cAMP dependent pathway. MC1R polymorphisms can impair this process, resulting in a predominance of red phaeomelanin. The red hair, fair skin and UV sensitive phenotype is a well-described melanoma risk factor. MC1R polymorphisms also confer melanoma risk independent of pigment. We investigated the effect of Mc1r deficiency in a mouse model of UV-induced melanoma. C57BL/6-Mc1r+/+-HGF transgenic mice have a characteristic hyperpigmented black phenotype with extra-follicular dermal melanocytes located at the dermal/epidermal junction. UVB induces melanoma, independent of melanin pigmentation, but UVA-induced and spontaneous melanomas are dependent on black eumelanin. We crossed these mice with yellow C57BL/6-Mc1re/e animals which have a non-functional Mc1r and produce predominantly yellow phaeomelanin. Yellow C57BL/6-Mc1re/e-HGF mice produced no melanoma in response to UVR or spontaneously even though the HGF transgene and its receptor Met were expressed. Total melanin was less than in C57BL/6-Mc1r+/+-HGF mice, hyperpigmentation was not observed and there were few extra-follicular melanocytes. Thus, functional Mc1r was required for expression of the transgenic HGF phenotype. Heterozygous C57BL/6-Mc1re/+-HGF mice were black and hyperpigmented and, although extra-follicular melanocytes and skin melanin content were similar to C57BL/6-Mc1r+/+-HGF animals, they developed UV-induced and spontaneous melanomas with significantly less efficiency by all criteria. Thus, heterozygosity for Mc1r was sufficient to restore the transgenic HGF phenotype but insufficient to fully restore melanoma. We conclude that a previously unsuspected melanin-independent interaction between Mc1r and Met signaling pathways is required for HGF-dependent melanoma and postulate that this pathway is involved in human melanoma.
doi:10.1002/ijc.29050
PMCID: PMC4262613  PMID: 24975581
22.  Metabolic engineering of Escherichia coli to optimize melanin synthesis from glucose 
Background
Natural aromatic polymers, mainly melanins, have potential and current applications in the cosmetic, pharmaceutical and chemical industries. The biotechnological production of this class of compounds is based on tyrosinase-dependent conversion of L-tyrosine and other aromatic substrates into melanins. The purpose of this work was to apply metabolic engineering for generating Escherichia coli strains with the capacity to synthesize an aromatic polymer from a simple carbon source.
Results
The strategy was based on the expression in E. coli of the MutmelA gene from Rhizobium etli, encoding an improved mutant tyrosinase. To direct the carbon flow from central metabolism into the common aromatic and the L-tyrosine biosynthetic pathways, feedback inhibition resistant versions of key enzymes were expressed in strains lacking the sugar phosphotransferase system and TyrR repressor. The expressed tyrosinase consumed intracellular L-tyrosine, thus causing growth impairment in the engineered strains. To avoid this issue, a two phase production process was devised, where tyrosinase activity was controlled by the delayed addition of the cofactor Cu. Following this procedure, 3.22 g/L of melanin were produced in 120 h with glucose as carbon source. Analysis of produced melanin by Fourier transform infrared spectroscopy revealed similar characteristics to a pure eumelanin standard.
Conclusions
This is the first report of a process for producing melanin from a simple carbon source at grams level, having the potential for reducing production cost when compared to technologies employing L-tyrosine as raw material.
doi:10.1186/1475-2859-12-108
PMCID: PMC3842659  PMID: 24225202
Melanin; L-tyrosine; Tyrosinase; Metabolic engineering; Escherichia coli
23.  Is there any difference in the photobiological properties of melanins isolated from human blue and brown eyes? 
Investigations were carried out to determine whether the melanin present in the blue and brown eyes were eumelanin, the melanin present in black hair and dark skin, or pheomelanin, the melanin present in red hair and the skin of people with red hair. Our results showed that UV-visible irradiation of blue or brown eye melanin did not produce any superoxide. Irradiation of 51Cr-labelled Ehrlich ascites carcinoma cells in the presence of blue or brown eye melanin did not produce significant cell lysis. The electron spin resonance (ESR) signals of blue and brown eye melanins were very similar to those of eumelanin. Comparison of these findings with our previous results indicated that the blue and brown eye melanins are essentially eumelanin. The ESR signals further suggested that in the case of both blue and brown eye melanins the iris, ciliary body, choroid, and retinal pigment epithelium did not differ.
PMCID: PMC1041224  PMID: 2820463
24.  Factors affecting phaeomelanin production by a melanin-producing (mel) mutant of Vibrio cholerae. 
Infection and Immunity  1981;34(3):895-899.
In a previous study we isolated melanin-producing (mel) mutants of Vibrio cholerae and demonstrated that production of melanin during growth on solid media was stimulated by L-tyrosine and L-cysteine. In the studies reported here we analyzed factors that affected melanin production in liquid media and determined the abilities of radioactively labeled amino acids to serve as precursors for the formation of melanin by V. cholerae. Radioactivity from L-cysteine and from L-tyrosine was preferentially incorporated into partially purified melanin, providing further evidence for production of phaeomelanin by V. cholerae. Cuprous ions stimulated production of melanin by V. cholerae in defined liquid medium, but melanin formation was inhibited by high concentrations of L-cysteine or thiouracil. The inhibition of melanin formation by sulfhydryl compounds is most likely due to their ability to bind copper ions that are essential for tyrosinase activity.
PMCID: PMC350953  PMID: 7333674
25.  In vitro removal of toxic heavy metals by poly(γ-glutamic acid)-coated superparamagnetic nanoparticles 
Background:
Chelation therapy involving organic chelators for treatment of heavy metal intoxication can cause cardiac arrest, kidney overload, mineral deficiency, and anemia.
Methods:
In this study, superparamagnetic iron oxide nanoparticles (SPIONs) modified with an edible biopolymer poly(γ-glutamic acid) (PGA) were synthesized by coprecipitation method, characterized and evaluated for their removal efficiency of heavy metals from a metal solution, and simulated gastrointestinal fluid (SGIF).
Results:
Instrumental characterization of bare- and PGA-SPIONs revealed 7% coating of PGA on SPIONs with a spherical shape and an iron oxide spinel structure belonging to magnetite. The particle sizes as determined from transmission electron microscopy images were 8.5 and 11.7 nm for bare- and PGA-SPIONs, respectively, while the magnetization values were 70.3 and 61.5 emu/g. Upon coating with PGA, the zeta potentials were shifted from positive to negative at most of the environmental pH (3–8) and biological pH (1–8), implying good dispersion in aqueous suspension and favorable conditions for heavy metal removal. Batch studies showed rapid removal of lead and cadmium with the kinetic rates estimated by pseudo-second-order model being 0.212 and 0.424 g/mg·min, respectively. A maximum removal occurred in the pH range 4–8 in deionized water and 5–8 in SGIF corresponding to most gastrointestinal pH except for the stomach. Addition of different ionic strengths (0.001–1 M sodium acetate) and essential metals (Cu, Fe, Zn, Mg, Ca, and K) did not show any marked influence on lead removal by PGA-SPIONs, but significantly reduced the binding of cadmium. Compared to deionized water, the lead removal from SGIF was high at all pH with the Langmuir monolayer removal capacity being 98.70 mg/g for the former and 147.71 mg/g for the latter. However, a lower cadmium removal capacity was shown for SGIF (23.15 mg/g) than for deionized water (31.13 mg/g).
Conclusion:
These results suggest that PGA-SPIONs could be used as a metal chelator for clinical treatment of metal poisoning.
doi:10.2147/IJN.S34396
PMCID: PMC3420602  PMID: 22927758
superparamagnetic iron oxide nanoparticles; poly(γ-glutamic acid); heavy metals; chelation therapy; gastrointestinal pH; kinetics

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