[Effects of mutation in dopachrome tautomerase on melanosome maturation and anti-oxidative potential in cultured melanocytes].

Objective To investigate whether the mutation in dopachrome tautomerase (Dct) affects melanosome maturation and anti-oxidative potential in cultured melanocytes (MCs). Methods Slaty and melan-a MCs were derived from the skins of neonatal DctSlt and C57 BI/6J mice respectively. Their detailed melanosome structures were examined with a transmission electron microscopy (TEM) and their eu-melanin granules characterized by Fontana-Masson staining. Furthermore, the tyrosinase activity and three melanogenic proteins, I.e. , tyrosinase, tyrosinase-related protein 1 and Dct, were also measured with a spectrophotometery method or Western blot assay. The level of intraceilular reactive oxygen species (ROS) was monitored by 2, 7-dichlorofluorescin diacetate (DCF-DA) labeling. Results Mature stage Ⅳ melanosomes markedly decreased in slaty MCs under TEM. The brownish granules stained with Fontana-Masson silver method were far less in slaty MCs than in melan-a MCs. The cell pellet of slaty MCs was white in color, but the similarities between slaty and melan-a were found in tyrosinase activity and its protein expression. The relative intensity of DCF fluorescence was 8.9±0.7 for slaty melanocytes versus 8.9±2.5 for melan-a melanocytes prior to UVA irradiation, but an abrupt ROS production was merely observed in slaty MCs (18.0±0.3) other than in melan-a MCs (13.6±0.3) after UVA exposure. There was statistical difference between these two cell lines in ROS level upon UVA irradiation (P=0.024). Conclusion The mutation in Det causes hypo-pigmented phenotype in cultured slaty MCs, inhibits melanosome maturation and decreases anti-oxidative capacity especially in the presence of UVA-induced oxidative stress. Key words: Isomerases;  Oxidative stress;  Mutation;  Melanosome