A combination of techniques, including high-performance liquid chromatography (HPLC), spectrophotometric measurements, and a novel method for quantifying melanosome morphology, were applied to the analysis of melanin content and composition in highly pigmented (Fitzpatrick type V and VI) human skin. We found that total epidermal melanin content is significantly elevated in photoexposed type V and VI skin (approximately 1.6 x), while analysis of individual melanin components suggests that pheomelanin content increases only slightly, whereas 5,6-dihydroxyindole-2-carboxylic acid (DHICA)-eumelanin and to a greater extent 5,6-dihydroxyindole (DHI)-eumelanin content are both markedly elevated. Analysis of the relative composition of epidermal melanin in these subjects revealed that DHI-eumelanin is the largest single component (approximately 60-70%), followed by DHICA-eumelanin (25-35%), with pheomelanin being a relatively minor component (2-8%). Moreover, there was a comparative enrichment of DHI-eumelanin at photoexposed sites, with a corresponding decline in the relative contributions from DHICA-eumelanin and pheomelanin. There was also a good correlation and close agreement between the concentration of spheroidal melanosomes determined by morphological image analysis and the concentration of pheomelanin determined by a combination of HPLC and spectrophotometric analysis (r = 0.89, P < 0.02). This study demonstrates the usefulness of melanosome morphology analysis as a sensitive new method for the quantification of melanin composition in human skin. The data also suggest that DHI-eumelanin formation is the dominant pathway for melanin synthesis in heavily pigmented (Fitzpatrick V and VI) skin types in vivo, and is the favoured pathway when melanin production is increased in chronically photoexposed skin.
We investigated the effect of varying concentration of 1-tyrosine and 1-cysteine in culture medium on melanin production by human skin melanocytes (skin phototype II/III). In addition to the analyses of dopa oxidase activity and total melanin, pheomelanin production in the cells was assessed by high-performance liquid chromatography determinations of pheomelanin degradation products, 3-aminotyrosine and 4-amino-3-hydroxyphenylalanine. As another marker for pheomelanin, melanosomal sulfur was determined by the use of X-ray microanalysis. With varying concentration of both amino acids, profound changes in the pigmentation patterns of the melanocytes were observed. A high concentration of 1-tyrosine (0.2 mM) was always connected with increased pigmentation. In combination with a low 1-cysteine content we saw an increase in tyrosinase activity and the highest melanin content. At high concentrations of both 1-tyrosine and 1-cysteine, the melanocytes showed reduced tyrosinase activity and they produced notably more pheomelanin. In case of the pheomelanin measurements by high-performance liquid chromatography and the sulfur detection with X-ray microanalysis, strongly increased concentrations were found when cells were maintained in high 1-tyrosine medium as compared with those grown with low 1-tyrosine. This was especially true for the combination with low 1-cysteine showing that the 1-tyrosine content of the medium strongly influences not only the eumelanin but also the pheomelanin production in the cultured melanocyte. It can be concluded that variations in the concentrations of 1-tyrosine and 1-cysteine in culture medium can be used to regulate the melanogenetic phenotype under in vitro conditions.
The goal of this investigation was to correlate the melanin content in human pigmentary cells with the generation of UVB-induced photoproducts and to examine the relationship between the melanin content and the removal of the photoproducts. Cultured melanocytes from light-skinned individuals synthesized less melanin and produced more cyclobutane pyrimidine dimers and 6-4 photoproducts upon UVB exposure than did melanocytes from black skin. Tyrosine-stimulated melanogenesis provided protection against DNA damage in both cell types. In another set of pigmented cell lines a ratio between eumelanin and pheomelanin was determined. The assessment of association between DNA damage induction and the quantity and quality of melanin revealed that eumelanin concentration correlated better with DNA protection than pheomelanin. Skin type-I and skin type-VI melanocytes, congenital nevus (CN)-derived cells and skin type-II melanocytes from a multiple-melanoma patient were grown in media with low or high L-tyrosine concentration. The cells were irradiated with 200 J/m2 UVB, and the levels of the photoproducts were determined immediately and after 6 and 24 h. Once again the induction of the photoproducts was mitigated by increased melanogenesis, and it was inversely correlated with the skin type. No significant differences were found for the removal of photoproducts in the cultures of skin types I and VI and CN cells. No indications of a delay in the removal of photoproducts in the melanocytes from the multiple-melanoma patient were found either.
The question of whether melanins are photoprotecting and/or photosensitizing in human skin cells continues to be debated. To evaluate the role of melanin upon UVA irradiation, DNA single-strand breaks (ssb) were measured in human melanocytes differing only in the amount of pigment produced by culturing at two different concentrations, basic (0.01 mM) or high (0.2 mM), of L-tyrosine, the main precursor of melanin. In parallel, pheo- and total melanin contents of the cells were determined. Identical experiments were performed with two melanocyte cultures derived from a skin type I and a skin type VI individual. For the first time the correlation between UVA-induced genotoxicity and pheo-/total melanin content has been investigated. We observed that cultured in basic medium, the skin type VI melanocytes contained 10 times more total melanin and about seven times more pheomelanin than the skin type I melanocytes. Elevation of tyrosine level in the culture medium resulted in an increase of both pheo- and total melanin levels in both melanocyte cultures; however, the melanin composition of skin type I melanocytes became more pheomelanogenic, whereas that of skin type VI melanocytes remained the same. The skin type VI melanocytes cultured in basic medium demonstrated a very high sensitivity (1.18 ssb per 10(10) Da per kJ per m2) toward UVA that is probably related to their high pheo- and total melanin content. Their UVA sensitivity, however, did not change after increasing their melanin content by culturing at high tyrosine concentration. In contrast, the skin type I melanocytes demonstrated a low sensitivity (0.04 ssb per 10(10) Da per kJ per m2) toward UVA when cultured in basic medium, but increasing their melanin content resulted in a 3-fold increase in their UVA sensitivity (0.13 ssb per 10(10) Da per kJ per m2). These results demonstrate that UVA-irradiated cultured human melanocytes are photosensitized by their own synthesized chromophores, most likely pheomelanin and/or melanin intermediates.
In many laboratories, culturing skin melanocytes has become a routine research activity. However, recent investigations have revealed that the quality and quantity of the pigment formed in the cultured cells may differ significantly from those of the original skin pigment cells. To shed more light on this issue, we examined the influence of different culture media on pigment production. We showed that there were notable passage-to-passage variations in the synthesis of melanin. This was particularly true for phaeomelanin. It is therefore advisable to analyse the melanin in the cells before the start of experiments. In spite of the variations, basic differences in the pigmentation pattern between melanocytes isolated from light-skinned and dark-skinned individuals remained preserved in the corresponding cultures as observed by electron microscopy. Also, the total melanin content was higher in a skin type VI melanocyte culture than in skin type I and II melanocyte cultures. In contrast to total melanin, the phaeomelanin concentration of skin type VI cells was similar to that of the skin type I melanocytes. With higher L-tyrosine concentrations in the medium, as well as increased eumelanin synthesis, phaeomelanogenesis was also stimulated in all cultures tested. This stimulation was particularly prominent in skin type I melanocytes. Our preliminary experiments also showed that a melanocyte culture from atypical naevus cells exhibited a similar preference for phaeomelanogenesis when pigmentation was stimulated.
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