[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 Dct(Slt) and C57 BL/6J mice respectively. Their detailed melanosome structures were examined with a transmission electron microscopy (TEM) and their eumelanin 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 spectrophotometry method or Western blot assay. The level of intracellular 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).


CONCLUSIONS
The mutation in Dct 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.
Wan
Jing
J
Department of Dermatology, Renmin Hospital of Wuhan University, Wuhan 430060, China.
Liu
Xiao-Ming
XM
Lei
Tie-Chi
TC
Xu
Shi-Zheng
SZ
D000818:Animals; D002478:Cells, Cultured; D019746:Intramolecular Oxidoreductases / Q000235:genetics*; D008543:Melanins / Q000096:biosynthesis; D008544:Melanocytes / Q000166:cytology; D020460:Melanosomes / Q000378:metabolism* / Q000648:ultrastructure; D051379:Mice; D008810:Mice, Inbred C57BL; D009154:Mutation; D018384:Oxidative Stress
D008543:Melanins; D019746:Intramolecular Oxidoreductases; C042501:dopachrome isomerase