Unraveling UVA1-Induced Photomodifications of Eumelanin and Pheomelanin in Human Skin: Insights into Pigment Darkening

UVA exposure elicits immediate and persistent pigment darkening of the skin, which is thought to result from the oxidation and polymerization of existing melanin and/or precursors. Melanocytes produce eumelanin and pheomelanin. Eumelanin consists of 5,6-dihydroxyindole (DHI) and 5,6-dihydroxyindole-2-carboxylic acid (DHICA), while pheomelanin consists of benzothiazine and benzothiazole units. Melanins can be analyzed by quantifying specific degradation products using HPLC. Specifically, eumelanin can be analyzed as pyrrole-2,3,5-tricarboxylic acid (PTCA) and pyrrole-2,3-dicarboxylic acid (PDCA), specific degradation products of DHICA and DHI, respectively. Benzothiazole pheomelanin can be analyzed as thiazole-2,4,5-tricarboxylic acid (TTCA), whereas benzothiazine pheomelanin is analyzed as 4-amino-3-hydroxyphenylalanine (4-AHP) and 3-amino-4-hydroxyphenylalanine (3-AHP). Upon UVA exposure, melanins undergo structural modifications. Eumelanin undergoes oxidative cleavage to free pyrrole-2,3,5-tricarboxylic acid (Free PTCA) and undergoes cross-linking to form pyrrole-2,3,4,5-tetracarboxylic acid (PTeCA). UVA exposure of pheomelanin induces oxidative conversion from the benzothiazine to the benzothiazole. Nevertheless, these structural modifications have never been previously characterized in human skin. In this study, we exposed ex vivo skin to increasing UVA1 doses (60, 90 and 120 J/cm2; n = 6 in triplicate) and characterized the induced pigment darkening before, immediately, and 2 h after exposure through colorimetry, HPLC and spectrophotometry. The results showed changes in the CIELAB colorimetric parameters, namely a decrease in Luminance L*, the yellow-blue component b* and the Individual Typology Angle (ITA) in UVA1-exposed samples, indicative of skin darkening. In parallel, UVA1 exposure induced significant modifications of the levels of absorbance at 500 nm (A500) and melanin markers PTCA, PTeCA, PDCA, TTCA, and 4-AHP, as well as in the ratios of various markers, such as PTeCA/PTCA, Free/Total PTCA, and TTCA/4-AHP, indicative of photooxidation/degradation of melanins. Our study provides the first evidence of UVA1-induced modifications of melanins associated with pigment darkening occurring in human skin.