Chemical Characterization of Melanins in Sheep Wool and Human Hair

The color of hair and wool in mammals and feathers in birds is mostly determined by the quantity and quality of melanins that are synthesized in follicular melanocytes and transferred to keratinocytes. There are two chemically distinct types of melanin pigments: the black to brown eumelanins and the yellow to reddish pheomelanins. Melanins in sheep wool and human hair of various colors were characterized by HPLC methods to estimate 5,6-dihydroxyindole-2-carboxylic acid (DHICA)-derived units in eumelanins and benzothiazine units in pheomelanins. Melanins were also characterized by spectrophotometric methods after differential solubilization in alkalies. It was demonstrated that 1) black wool in Asiatic sheep contains eumelanin with the DHICA content similar to black mouse melanin, while black to brown melanins from human hair contain much lower ratios of DHICA-derived units, comparable to the slaty mutation in mice, 2) dark brown to brown hair in human contains eumelanin whose chemical properties are indistinguishable from those of black hair, 3) dark red wool and red human hair contain pheomelanic pigments whose chemical properties are rather different from those of yellow pheomelanins in mice, and 4) light brown, blonde, and red hairs in human can be differentiated from each other with this methodology.     

Enzymatic control of pigmentation in mammals.

Visible pigmentation in mammals results from the synthesis and distribution of melanin in the skin, hair bulbs, and eyes. The melanins are produced in melanocytes and can be of two basic types: eumelanins, which are brown or black, and phaseomelanins, which are red or yellow. In mammals typically there are mixtures of both types. The most essential enzyme in this melanin biosynthetic pathway is tyrosinase and it is the only enzyme absolutely required for melanin production. However, recent studies have shown that mammalian melanogenesis is not regulated solely by tyrosinase at the enzymatic level, and have identified additional melanogenic factors that can modulate pigmentation in either a positive or negative fashion. In addition, other pigment-specific genes that are related to tyrosinase have been cloned which encode proteins that apparently work together at the catalytic level to specify the quantity and quality of the melanins synthesized. Future research should provide a greater understanding of the enzymatic interactions, processing, and tissue specificity that are important to pigmentation in mammals.     

肤色、黑色素皮质素受体1和紫外线

近期的研究表明,哺乳动物黑素细胞中黑色素皮质素受体1（MC1R）对调节棕黑色素和红黄色素的合成起关键的作用。MC1R基因的变异与动物的皮毛、人的皮肤和头发颜色差异密切相关。对小鼠的遗传学研究显示,MC1R是独特的、双功能控制受体。它由α-促黑色素皮质激素激活,其拮抗物为agouti蛋白,二者的共同作用导致哺乳动物表皮颜色的变异。另外,人类皮肤的色素沉着是决定于皮肤对外辐射的反应,以及由此引发皮肤癌的重要因素。MC1R变异与黑色素癌易感性相关。 Genotype,Melanocortin 1 Receptor and Ultrviolet Radiation Lü Xue-mei,SHI Peng,ZHANG Ya-ping Lab of Cellular & Molecular Evolution,Kunming Institute of Zoology,the Chinese Academy of Sciences,Kunming 650223,China Abstract:Recent work on the melanocortin 1 receptor (MC1R) suggests that MC1R plays a central role in regulation of eumelanin (brwon/black melanins) and phaeomelanin (red/yellow melanins) synthesis within the mammalian melanocyte.In the mouse,genetic studies show that the MC1R appears to be a unique,bifunctionally controlled receptor,activated by α–MSH and antagonized by agouti,both of which contribute to the variability seen in mammalian coat color.Variants of this receptor are associated with different animal's coat,human skin and hair colors.In addition,cutaneous pigmentation is a major determinant of the cutaneous response to ultraviolet radiation,and consequently of the risk of developing skin cancer.MC1R variants are a risk factor for melanoma susceptibility. Key words：Melanocortin-1 receptor gene; MC1R variants; ultraviolet radiation; skin and hair colors; skin cancer     

Melanins and melanosomes from llama (Lama glama L.)

Analysis of melanins and melanosomes in eight hair and skin samples taken of adult pigmented Argentine llamas (Lama glama L.) has been carried out. In each sample, eumelanins, pheomelanins and alkali-soluble melanins were identified. The total amount of melanins and the amount of eumelanins both decreased from black to reddish brown colour, while pheomelanins were found to be present in small quantities in each sample. Eumelanosomes were round and oval-shaped, displaying transverse striations clearly visible at low magnification. Dark brown samples revealed all four melanosomes stages. Stages I and II melanosomes appeared as large, asymmetrical vacuoles containing numerous microvesicles randomly scattered within an amorphous proteinaceous material (vesiculo-globular bodies). Stage III melanosomes had microgranular melanin deposits in the microvesicles and in the matrix. The fully melanized melanosomes (stage IV) were primarily round-shaped, showing an irregular outline and the electron-dense pigment was arranged to form large clusters. In light brown melanocytes, numerous melanosomes at different maturation stages could be found. Premelanosomes appeared ovoid, containing amorphous proteinaceous material and spotty and microgranular deposits. Mature melanosomes were fully melanized, homogeneously electron-dense, ovoid granules.     

Eumelanin and Phaeomelanin Contents of Human Epidermis and Cultured Melanocytes

There are two chemically distinct types of melanin: the red-yellow phaeomelanins and the brown-black eumelanins. While both melanins have been detected in human epidermis and cultured melanocytes, it is unknown how the phaeomelanin/eumelanin ratio in human melanocytes maintained in vitro relates to that in the epidermis from which they were isolated. This study uses high-performance liquid chromatography to quantify the eumelanin and phaeomelanin contents of epidermis and/or cultured melanocytes from 12 Europeans with lightly pigmented skin and 9 non-Europeans with more deeply pigmented skin. Epidermis from non-Europeans contained the highest levels of both eumelanin and phaeomelanin and had the lowest phaeomelanin/eumelanin ratios. In contrast, while cultured melanocytes from non-Europeans also had higher levels of eumelanin and phaeomelanin than melanocytes from Europeans, there was no difference in the phaeomelanin/eumelanin ratios in the two groups. However, the phaeomelanin/eumelanin ratios were higher in the cultured melanocytes than in the corresponding epidermis so that while eumelanin was the predominant melanin in the epidermis, phaeomelanin was the major melanin in the cultured melanocytes. These observations may have important implications for the use of cultured human melanocytes in the study of melanogenesis in man.     

MC1R and the response of melanocytes to ultraviolet radiation

The constitutive color of our skin plays a dramatic role in our photoprotection from solar ultraviolet radiation (UVR) that reaches the Earth and in minimizing DNA damage that gives rise to skin cancer. More than 120 genes have been identified and shown to regulate pigmentation, one of the key genes being melanocortin 1 receptor (MC1R) that encodes the melanocortin 1 receptor (MC1R), a seven-transmembrane G protein-coupled receptor expressed on the surface of melanocytes. Modulation of MC1R function regulates melanin synthesis by melanocytes qualitatively and quantitatively. The MC1R is regulated by the physiological agonists alpha-melanocyte-stimulating hormone (alphaMSH) and adrenocorticotropic hormone (ACTH), and antagonist agouti signaling protein (ASP). Activation of the MC1R by binding of an agonist stimulates the synthesis of eumelanin primarily via activation of adenylate cyclase. The significance of cutaneous pigmentation lies in the photoprotective effect of melanin, particularly eumelanin, against sun-induced carcinogenesis. Epidermal melanocytes and keratinocytes respond to UVR by increasing their expression of alphaMSH and ACTH, which up-regulate the expression of MC1R, and consequently enhance the response of melanocytes to melanocortins. Constitutive skin pigmentation dramatically affects the incidence of skin cancer. The pigmentary phenotype characterized by red hair, fair complexion, inability to tan and tendency to freckle is an independent risk factor for all skin cancers, including melanoma. The MC1R gene is highly polymorphic in human populations, and allelic variation at this locus accounts, to a large extent, for the variation in pigmentary phenotypes and skin phototypes (SPT) in humans. Several allelic variants of the MC1R gene are associated with the red hair and fair skin (RHC) phenotype, and carrying one of these variants is thought to diminish the ability of the epidermis to respond to DNA damage elicited by UVR. The MC1R gene is considered a melanoma susceptibility gene, and its significance in determining the risk for skin cancer is of tremendous interest.     

High polymorphism at the human melanocortin 1 receptor locus

Variation in human skin/hair pigmentation is due to varied amounts of eumelanin (brown/black melanins) and phaeomelanin (red/yellow melanins) produced by the melanocytes. The melanocortin 1 receptor (MC1R) is a regulator of eu- and phaeomelanin production in the melanocytes, and MC1R mutations causing coat color changes are known in many mammals. We have sequenced the MC1R gene in 121 individuals sampled from world populations with an emphasis on Asian populations. We found variation at five nonsynonymous sites (resulting in the variants Arg67Gln, Asp84Glu, Val92Met, Arg151Cys, and Arg163Gln), but at only one synonymous site (A942G). Interestingly, the human consensus protein sequence is observed in all 25 African individuals studied, but at lower frequencies in the other populations examined, especially in East and Southeast Asians. The Arg163Gln variant is absent in the Africans studied, almost absent in Europeans, and at a low frequency (7%) in Indians, but is at an exceptionally high frequency (70%) in East and Southeast Asians. The MC1R gene in common and pygmy chimpanzees, gorilla, orangutan, and baboon was sequenced to study the evolution of MC1R. The ancestral human MC1R sequence is identical to the human consensus protein sequence, while MC1R varies considerably among higher primates. A comparison of the rates of substitution in genes in the melanocortin receptor family indicates that MC1R has evolved the fastest. In addition, the nucleotide diversity at the MC1R locus is shown to be several times higher than the average nucleotide diversity in human populations, possibly due to diversifying selection.     

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.     

Chemical and Electron Microscopic Studies of Cattle (Bos taurus) With Four Types of Phenotypic Pigmentation

The biological behavior of the pigmentary phenotypes of four breeds of cattle has been analysed: the black pigmentation of Holstein Friesian; the red pigmentation of Limousin; the dilution in Charolais; and the postnatal disappearance of red pigmentation in Chianina. The analytic techniques included the characterization of melanins by high-performance liquid chromatography, the examination of follicular melanocytes by light microscopy, and the examination of melanosomes by electron microscopy. The black phenotype was very strongly eumelanogenic. The red phenotype in Limousin is polymorphic: individual follicular melanocytes contain both mature eumelanosomes and pheomelanosomes. Charolais and Chianina cattle exhibited a dramatic reduction in melanogenic activity, which was characterized by the almost exclusive presence of prephaoemelanosomes in Charolais and of immature premelanosomes in Chianina. In the dilute Charolais phenotype, the density of distribution of follicular melanocytes also seemed to be reduced. The genes that are responsible for these four phenotypes seem to act on the maturation, differentiation, and density of distribution of the melanosomes.     

OPRM1 and EGFR contribute to skin pigmentation differences between Indigenous Americans and Europeans

Contemporary variation in skin pigmentation is the result of hundreds of thousands years of human evolution in new and changing environments. Previous studies have identified several genes involved in skin pigmentation differences among African, Asian, and European populations. However, none have examined skin pigmentation variation among Indigenous American populations, creating a critical gap in our understanding of skin pigmentation variation. This study investigates signatures of selection at 76 pigmentation candidate genes that may contribute to skin pigmentation differences between Indigenous Americans and Europeans. Analysis was performed on two samples of Indigenous Americans genotyped on genome-wide SNP arrays. Using four tests for natural selection--locus-specific branch length (LSBL), ratio of heterozygosities (lnRH), Tajima's D difference, and extended haplotype homozygosity (EHH)--we identified 14 selection-nominated candidate genes (SNCGs). SNPs in each of the SNCGs were tested for association with skin pigmentation in 515 admixed Indigenous American and European individuals from regions of the Americas with high ground-level ultraviolet radiation. In addition to SLC24A5 and SLC45A2, genes previously associated with European/non-European differences in skin pigmentation, OPRM1 and EGFR were associated with variation in skin pigmentation in New World populations for the first time.     

Application of Photoacoustic Phase Angle Spectroscopy (φAS) to Eumelanins and Pheomelanins

A new technique, based on the measurement of the phase angle of the photoacoustic signal, was used for investigating the absorption spectra of melanins in the dry state. The main advantages of such a method are the insensitivity to scattering of the light and the applicability to nontransparent or highly adsorbing and thermally thick samples. The spectra obtained on different kinds of natural and synthetic melanins show much more details than the corresponding ones obtained spectrophotometrically on aqueous suspensions of the pigment. Some relevant features of the spectra still cannot be interpreted, yet we were able to show the presence in eumelanins of dopachrome at 475 nm and melanochrome at 540 nm, and in pheomelanins of compounds previously isolated from red chicken feathers. Moreover, the well known trend of the optical absorption, i.e., its decrease with increasing wavelength, was unambiguously confirmed.     

Was skin cancer a selective force for black pigmentation in early hominin evolution?

Melanin provides a crucial filter for solar UV radiation and its genetically determined variation influences both skin pigmentation and risk of cancer. Genetic evidence suggests that the acquisition of a highly stable melanocortin 1 receptor allele promoting black pigmentation arose around the time of savannah colonization by hominins at some 1–2 Ma. The adaptive significance of dark skin is generally believed to be protection from UV damage but the pathologies that might have had a deleterious impact on survival and/or reproductive fitness, though much debated, are uncertain. Here, I suggest that data on age-associated cancer incidence and lethality in albinos living at low latitudes in both Africa and Central America support the contention that skin cancer could have provided a potent selective force for the emergence of black skin in early hominins.     

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.     

The Interaction of Agouti Signal Protein and Melanocyte Stimulating Hormone to Regulate Melanin Formation in Mammals

Important regulatory controls of melanogenesis that operate at the subcellular level to modulate the structural and/or the functional nature of the melanins and melanin granules produced in melanocytes are reviewed. Melanocyte stimulating hormone and agouti signal protein have antagonistic roles and possibly opposing mechanisms of action in the melanocyte. In the mouse, melanocyte stimulating hormone promotes melanogenic enzyme function and elicits increases in the amount of eumelanins produced, while agouti signal protein reduces total melanin production and elicits the synthesis of pheomelanin rather than eumelanin. We are now beginning to understand the complex controls involved in regulating this switch at the molecular and biochemical levels. The quality and quantity of melanins produced by melanocytes have important physiological consequences for melanocyte function and undoubtedly play important roles in the various functions of the melanins per se, including hair and skin coloration and photoprotection.     

Chemical analysis of melanin pigments in feather germs of Japanese quail Bh (black at hatch) mutants.

Bh (black at hatch) is a mutation of Japanese quails which causes darkening or lightening of the plumage in heterozygotes or homozygotes, respectively. We chemically analyzed melanin pigments in feather germs of Bh mutant embryos and in feathers of adult animals. Dark brown dorsal feathers of wild-type adult animals had white barrings, but heterozygous ones lacked clear barrings. The feathers of wild-type and heterozygote animals contained both eumelanins and pheomelanins, the latter being more pheomelanic. On the dorsal skin of 10-day old wild-type embryos, longitudinal stripes from black and yellow rows of feather germs developed; two or three longitudinal rows of black feather germs and then two or three rows of yellow feather germs next to the short central feather germs. Heterozygous embryos appeared black in plumage pigmentation, due to the presence of 'gray' feather germs in rows of dorsal feather germs that corresponded to yellow rows in wild-type embryos. Homozygous dorsal feather germs did not develop the black and yellow longitudinal stripes, but were brown. Chemical analysis showed that embryos of each genotype contained both eumelanins and pheomelanins in the feather germs; however, the eumelanin content in homozygous feather germs was very low. These results suggest that the Bh mutation causes pheomelanic changes in feathers of quails.     

UVA-induced oxidative degradation of melanins: fission of indole moiety in eumelanin and conversion to benzothiazole moiety in pheomelanin

Eumelanin is photoprotective while pheomelanin is phototoxic to pigmented tissues. Ultraviolet A (UVA)-induced tanning seems to result from the photooxidation of pre-existing melanin and contributes no photoprotection. However, data available for melanin biodegradation remain limited. In this study, we first examined photodegradation of eumelanin and pheomelanin in human black hairs and found that the ratio of Free (formed by peroxidation in situ) to Total (after hydrogen peroxide oxidation) pyrrole-2,3,5-tricarboxylic acid (PTCA) increases with hair aging, indicating fission of the dihydroxyindole moiety. In red hair, the ratio of thiazole-2,4,5-tricarboxylic acid (TTCA) to 4-amino-3-hydroxyphenylalanine (4-AHP) increases with aging, indicating the conversion from benzothiazine to benzothiazole moiety. These photodegradation of melanins were confirmed by UVA (not UVB) irradiation of melanins from mice and human hairs and synthetic eumelanin and pheomelanin. These results show that both eumelanin and pheomelanin degrade by UVA and that Free/Total PTCA and TTCA/4-AHP ratios serve as sensitive indicators of photodegradation.     

Chemical Analysis of Melanin Pigments in Feather Germs of Japanese Quail Bh (Black at Hatch) Mutants

Bh (black at hatch) is a mutation of Japanese quails which causes darkening or lightening of the plumage in heterozygotes or homozygotes, respectively. We chemically analyzed melanin pigments in feather germs of Bh mutant embryos and in feathers of adult animals. Dark brown dorsal feathers of wild-type adult animals had white barrings, but heterozygous ones lacked clear barrings. The feathers of wild-type and heterozygote animals contained both eumelanins and pheomelanins, the latter being more pheomelanic. On the dorsal skin of 10-day old wild-type embryos, longitudinal stripes from black and yellow rows of feather germs developed; two or three longitudinal rows of black feather germs and then two or three rows of yellow feather germs next to the short central feather germs. Heterozygous embryos appeared black in plumage pigmentation, due to the presence of‘gray’feather germs in rows of dorsal feather germs that corresponded to yellow rows in wild-type embryos. Homozygous dorsal feather germs did not develop the black and yellow longitudinal stripes, but were brown. Chemical analysis showed that embryos of each genotype contained both eumelanins and pheomelanins in the feather germs; however, the eumelanin content in ho-mozygous feather germs was very low. These results suggest that the Bh mutation causes pheomelanic changes in feathers of quails.     

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.     

Childhood exposure to ultraviolet radiation and harmful skin effects: epidemiological evidence

We review the general amount and patterns of exposure to solar ultraviolet (UV) radiation that children and teenagers experience and the spectrum of UV-related skin damage that can occur as a result. Data about the amount of solar UV received by children and teenagers are relatively few but suggest that around 40–50% of total UV to age 60 occurs before age 20. Among white children, those with the palest complexions suffer the most damage. Comparisons of prevalence and incidence of outcomes in children and teenagers sharing common ancestry, but living at different latitudes, show that prevalence rates of photoaging and melanocytic naevi are higher in Australian compared with British children, and similarly for melanoma. Genetic risk for the majority of the melanomas in teens is a function of genes controlling naevus propensity and pigmentation in the skin. High numbers of naevi and freckles, red hair, blue eyes, inability to tan, as well as a family history are the primary determinants of melanoma among adolescents. Beyond the signs of skin damage seen in children are the latent effects observed later in adulthood. Childhood is believed to be a susceptible window for long-term harmful effects of UV, as evidenced by clear differences in skin cancer risk between child and adult migrants from high to low latitudes. Effective UV radiation protection from childhood is necessary to control both immediate and long-term harmful effects on children’s skin.     

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.