Oxidation Levels Differentially Impact Melanocytes: Low versus High Concentration of Hydrogen Peroxide Promotes Melanin Synthesis and Melanosome Transfer

Abstract

Background: UVB light can generate potentially harmful hydrogen peroxide (H₂O₂) in vivo, but it can also promote the beneficial proliferation and migration of melanocytes. The successful use of UVB monotherapy for treatment of vitiligo suggests that H₂O₂ may have a biphasic effect on melanin synthesis and melanosome transfer. Objective: To study the beneficial role of H₂O₂ on melanogenesis and melanosome transport in living melanocytes and keratinocytes. Methods: A co-culture system model was constructed using the primary human melanocytes and keratinocytes. The MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay was used to determine cell proliferation, NaOH was used to determine the melanin content, and real-time PCR was used to determine tyrosinase expression. Western blot was used to determine Rab-27A and protease-activated receptor 2 (PAR-2) expression. Results: This study demonstrated that tyrosinase was activated by low concentrations of H₂O₂ (≤0.3 mM); however, this activity was downregulated by high concentrations of H₂O₂ (>0.3 mM). Activation of high levels of melanin synthesis was induced when cells were treated with low concentrations of H₂O₂ (0.3 mM). Further observation using an in vitro co-culture system of fluorescein (carboxyfluorescein diacetate succinimidyl ester, CFDA-SE)-labeled melanocytes and keratinocytes indicated that melanosome transfer occurred in normal human epidermal melanocytes. Fluorescence microscopy revealed increased melanosome transfer into keratinocytes treated with 0.3 mM H₂O₂ in the co-culture compared to the control. Examination of melanosomes in the keratinocytes by flow cytometry confirmed these results. Furthermore, treatment with H₂O₂ (0.3 mM) upregulated the expression of Rab-27A and PAR-2, significant proteins involved in melanosome transfer, according to Western blot. Conclusion: These results confirmed that low concentration levels of H₂O₂ play a major role in the regulation of human pigmentation by increasing melanin synthesis and melanosome transfer.