5,6-Dihydroxyindole eumelanin content in human skin with varying degrees of constitutive pigmentation.

Human skin contains two distinct components: brown to black, insoluble eumelanin and light colored, alkaline-soluble pheomelanin. Eumelanin consists of 5,6-dihydroxyindole (DHI) and 5,6-dihydroxyindole-2-carboxylic acid (DHICA) moieties, while pheomelanin consists of benzothiazine (BT) and benzothiazole (BZ) moieties. These melanin monomer units can be quantitatively analyzed through specific degradation products by high-performance liquid chromatography (HPLC). Alkaline hydrogen peroxide oxidation (AHPO) of eumelanin gives rise to pyrrole-2,3,5-tricarboxylic acid (PTCA) and pyrrole-2,3-dicarboxylic acid (PDCA) as specific degradation products of the DHICA and DHI moieties, respectively. BZ moiety in pheomelanin can be analyzed as thiazole-2,4,5-tricarboxylic acid (TTCA). By reductive hydrolysis with hydroiodic acid, BT moieties in pheomelanin can be analyzed as 4-amino-3-hydroxyphenylalanine (4-AHP). As a recently improved AHPO-HPLC method enabled a better characterization of PDCA, this prompted us to address the question of DHI to DHICA ratio in human skin samples with varying degrees of constitutive pigmentation ranging from very light to dark. Results showed for the first time the ratio of 4 moieties: DHI 35%, DHICA 41%, BZ 20%, and BT 4%. The ratio is constant regardless of the degree of pigmentation. The high content of DHICA moiety may impart an antioxidant property to the epidermis melanin.

Chemical analysis of constitutive pigmentation of human epidermis reveals constant eumelanin to pheomelanin ratio.

The skin constitutive pigmentation is given by the amount of melanin pigment, its relative composition (eu/pheomelanin) and distribution within the epidermis, and is largely responsible for the sensitivity to UV exposure. Nevertheless, a precise knowledge of melanins in human skin is lacking. We characterized the melanin content of human breast skin samples with variable pigmentations rigorously classified through the Individual Typology Angle (ITA) by image analysis, spectrophotometry after solubilization with Soluene-350 and high-performance liquid chromatography (HPLC) after chemical degradation. ITA and total melanin content were found correlated, ITA and PTCA (degradation product of DHICA melanin), and TTCA (degradation product of benzothiazole-type pheomelanin) as well but not 4-AHP (degradation product of benzothiazine-type pheomelanin). Results revealed that human epidermis comprises approximately 74% of eumelanin and 26% pheomelanin, regardless of the degree of pigmentation. They also confirm the low content of photoprotective eumelanin among lighter skins thereby explaining the higher sensitivity toward UV exposure.

Different oxidative stress response in keratinocytes and fibroblasts of reconstructed skin exposed to non extreme daily-ultraviolet radiation.

Experiments characterizing the biological effects of sun exposure have usually involved solar simulators. However, they addressed the worst case scenario i.e. zenithal sun, rarely found in common outdoor activities. A non-extreme ultraviolet radiation (UV) spectrum referred as "daily UV radiation" (DUVR) with a higher UVA (320-400 nm) to UVB (280-320 nm) irradiance ratio has therefore been defined. In this study, the biological impact of an acute exposure to low physiological doses of DUVR (corresponding to 10 and 20% of the dose received per day in Paris mid-April) on a 3 dimensional reconstructed skin model, was analysed. In such conditions, epidermal and dermal morphological alterations could only be detected after the highest dose of DUVR. We then focused on oxidative stress response induced by DUVR, by analyzing the modulation of mRNA level of 24 markers in parallel in fibroblasts and keratinocytes. DUVR significantly modulated mRNA levels of these markers in both cell types. A cell type differential response was noticed: it was faster in fibroblasts, with a majority of inductions and high levels of modulation in contrast to keratinocyte response. Our results thus revealed a higher sensitivity in response to oxidative stress of dermal fibroblasts although located deeper in the skin, giving new insights into the skin biological events occurring in everyday UV exposure.