| Abstract: | Immediate pigment darkening (IPD), which is a skin tanning reaction induced by UVA or visible light irradiation, is generally thought to result from photo oxidation of pre-existing melanin. Only a few in vitro systems useful for studying the detailed mechanisms have been developed. The purpose of this study is to present UVA-induced darkening at the cell level in an in vitro system and clarify the mechanisms of IPD. Human lower epidermal cells (LECs) separated by the Percoll density gradient centrifugation and murine B16 malignant melanoma cells (B16Cs) were treated to prepare a melanin containing, pyridine-insoluble precipitate (PUP). The degree of darkness of the UVA-exposed cells was expressed as reflectance at 400 nm (RE) from PUP. With increasing UVA dose, the RE from B16C-PUP induced no significant change, but the RE from LEC-PUP decreased. Thus the model of UVA-induced darkening at the cell level using LEC was successful. Measurement of the height of electron spin resonance (ESR) signal from PUPs of whole epidermal cells and of B16Cs revealed a 10% decrease in the former and no significant change in the latter after UVA treatment. These results suggest that a combination of the melanin in intracellular melanosomes and UVA exposure alone did not induce melanin darkening of IPD, and also that the semiquinoid form of melanin monomeric units decreased in the keratinocyte melanin. Taking into consideration the molecular structure of melanin, these results also suggest that photo oxidation of melanin monomeric units from the semiquinoid to the quinoid form is one of the mechanisms of IPD causing the darkening of melanin pigment. |
