Fig 1.
Chemical structures of tyrosine, HQ, arbutin and D-Arb.
HQ, arbutin, and D-Arb share a core phenolic moiety with tyrosine in their chemical structures, as shown in red broken-line box.
Fig 2.
Phenotypic changes and melanin distribution in hyperpigmented guinea pig skin treated with HQ.
A: Hyperpigmentation in guinea pig dorsal skin was induced by 308-nm MEL radiation. Changes in skin pigmentation were observed at the irradiated sites treated with (c) 3% H2O2, (d) 5% HQ, (e) 10% arbutin or (f) 10% D-Arb, once a day for 10 days, as described under Materials and methods. (a) Sham-irradiated control and (b) vehicle control. B: Fontana-Masson staining reveals supranuclear melanin as black-brown granules in the basal and suprabasal layers in skins of the vehicle control (b). Less pronounced melanin deposits are seen in (d) 5% HQ or (f) 10% D-Arb. It is noted that HQ caused an increase in epidermal thickness (d). Scale bar, 50 μm.
Fig 3.
Measurement of melanin remnants in surface corneocytes using Fontana-Masson silver staining.
Surface corneocytes were collected from guinea pig dorsal skin treated as described in Fig 2. Many coarseclustered black silver deposits are seen in corneocytes from vehicle controls (b)and H2O2-treated group (c), whereas fewer silver precipitations are seen in the sham-irradiated control (a) andthe depigmented skin after topical application of 5% HQ (d), 10% arbutin (e) and 10% D-Arb (f) for 10 days. Scale bars: 20 μm.
Fig 4.
Ultrastructural observation of melanosomes within keratinocytes of hyperpigmented guinea pig skin treated with HQ.
The dorsal skins of guinea pigs were treated as described for Fig 2. Marked vacuolations and fragmentations of melanosomes are seen in the depigmented skin after topical application of 5% HQ for 10 days (d1 and d2, arrows). The outer membranes of melanosomes in skins from (a) sham-irradiated control, (b) vehicle control, and (f) 10% D-Arb seem to be intact or have minor damage. Low-magnification views (a1-f1)are shown in the upper panels, the white boxes mark the areas magnified in the panels below (a2-f2). Scale bars: 2 μm(low-); 0.5 μm (high-magnification).
Table 1.
Comparisons of epidermis thickness, L* value, damagedmelanosomes and melanin particles in skins treated with H2O2, HQor its derivatives.
Fig 5.
Ultrastructural observations of individual naked melanosomes.
A: Individual naked melanosomes were purified from cultured MNT1 cells, as described under Materials and methods. Typical images of mature melanosomes are shown in low (a) (5000×) and in higher (b) (15000×) magnifications. Melanosome fractions were treated with freeze-thawing (FT) plus manual grinding (c), 8M urea (d), 100 μM H2O2 (e), 3J/cm2 UVA radiation(f), 10 μM HQ (g) and 100 μM D-Arb (h). Significantly fragmented and vacuolated melanosomes are seen in specimens treated with 100 μM H2O2, 3 J/cm2 UVA radiation, and 10 μM HQ (e-g). Scale bar: 1μm (except 0.2 μm for b and 0.5 μm for e). B: Comparison of percentages ofdamaged melanosomes following the different treatments. Two-way ANOVA was used to determine the statistical difference between treated melanosomes and the untreated control. * P<0.05, # P> 0.05.
Fig 6.
Effects of melanosomes treated with HQ on hydroxyl radical generation in theFenton reaction.
A: Representative ESR spectra of DMPO–OH with melanosomes treated with 100 μM H2O2, 10 μM HQ or 100 μM D-Arb. Hydroxyl radicals are generated by the Fenton reaction (DMPO: 400 mM). B: Comparison of hydroxyl radical-scavenging activity for melanosomes with different treatments. Two-way ANOVA was used to determine the statistical difference between treated melanosomes and the untreated control. * P<0.05.