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Background Rhododendrol (RD) is cytotoxic to melanocytes and can cause leukoderma at the application site. Patients with RD-induced leukoderma usually recover after discontinuation of application (improved cases). However, occasionally lesions may expand or new lesions may develop in unapplied sites (aggravated cases). Objective To understand the pathomechanism of RD-induced leukoderma in aggravatedcases. Methods We performed immunohistochemical study to compare the expression of the catalytic subunit of glutamate cysteine ligase (GCLC), E-cadherin, and mast cell tryptase in 22 improved cases, 16 aggravated cases, and 39 normal controls. Results GCLC expression in melanocytes surrounding leukoderma was higher in improved cases than in normal controls and aggravated cases. E-cadherin expression in melanocytes was upregulated both in improved and aggravated cases and increased levels of degranulated mast cells were observed in aggravated cases. Conclusion Our results indicate that downregulation of GCLC expression due to low levels of glutathione along with reduced melanocyte migration and increased E-cadherin expression can aggravate RD-induced leukoderma. Additionally, increased degranulation of mast cells may prolong the persistence of RD-induced leukoderma.

Background Rhododendrol, 4-(4-hydroxyphenyl)-2-butanol, Rhododenol® (RD), a naturally occurring phenolic compound, was developed as a tyrosinase inhibitor for skin-lightening/whitening cosmetics. In 2013, skin depigmentation was reported in consumers using RD-containing skin-brightening cosmetics; this condition is called RD-induced leukoderma. Objective The etiology of RD-induced leukoderma is still largely unknown. Here, to assess the depigmentation potential of RD, we developed a new mouse model of leukoderma by topically applying RD. Methods Hairless hk14-SCF Tg mice with melanocytes distributed in the epidermis were used for this study. RD was applied on the dorsal skin of the mice daily for 28 days. Then, immunohistological, biochemical, and electron microscopic analyses were performed on biopsy samples taken from these mice. Results The depigmentation in the RD-treated sites appeared on Day 14. Histological examination indicated a loss of epidermal melanocytes at Day 7. On the other hand, the melanocyte number did not decrease in the albino mice having the same background as the hairless hk14-SCF Tg, but without tyrosinase activity. Biochemical analyses showed that the eumelanin content decreased in the RD-treated sites and metabolites of RD-quinone, i.e., non-protein thiol adducts and protein-SH adducts, were produced. Electron microscopic analyses revealed double-membrane-walled structures containing electron-dense material, which might be typical for melanin-containing autophagosomes and a dilated endoplasmic reticulum (ER), which would indicate ER stress. Conclusions These data suggested that RD exerted tyrosinase-dependent melanocyte cytotoxicity and that tyrosinase-dependent accumulation of ER stress from activation of the autophagy pathway contributed to melanocyte cytotoxicity.

Background Tyrosinase, the rate-limiting enzyme required for melanin production, has been targeted to develop active brightening/lightening materials for skin products. Unexpected depigmentation of the skin characterized with the diverse symptoms was reported in some subjects who used a tyrosinase-competitive inhibiting quasi-drug, rhododendrol. Objective To investigate the mechanism underlying the depigmentation caused by rhododendrol-containing cosmetics, this study was performed. Methods The mechanism above was examined using more than dozen of melanocytes derived from donors of different ethnic backgrounds. The RNAi technology was utilized to confirm the effect of tyrosinase to induce the cytotoxicity of rhododendrol and liquid chromatography-tandem mass spectrometry was introduced to detect rhododendrol and its metabolites in the presence of tyrosinase. Results Melanocyte damage was related to tyrosinase activity at a certain threshold. Treatment with a tyrosinase -specific siRNA was shown to dramatically rescue the rhododendrol-induced melanocyte impairment. Hydroxyl-rhododendrol was detected only in melanocytes with higher tyrosinase activity. When an equivalent amount of hydroxyl-rhododendrol was administered, cell viability was almost equally suppressed even in melanocytes with lower tyrosinase activity. Conclusion The generation of a tyrosinase-catalyzed hydroxyl-metabolite is one of the causes for the diminishment of the melanocyte viability by rhododendrol.

Background Oculocutaneous albinism (OCA) type 3 caused by mutations of the TYRP1 gene is an autosomal recessive disorder of pigmentation characterized by reduced biosynthesis of melanin pigment in the skin, hair, and eye. The clinical phenotype has been reported as mild in Caucasian OCA3 patients. Objective We had the opportunity to examine a Japanese girl with OCA3 and investigated activity of TYRP1 protein derived from the mutant allele detected in the patient. Methods Mutation search for OCA responsible genes was done. A mutant allele with a missense mutation was analyzed using melanocyte cultures (b cells) established from a mouse model of OCA3. Results Compound heterozygous mutations, p.C30R and p.367fsX384, were detected in the Japanese girl. Then we revealed that the missense mutation, p.C30R, was functionally incapable of melanin synthesis with in vitro experiments. Conclusion This is the first report of the occurrence of OCA3 in Japanese population.

Patients with OCA are characterized by reduced skin and hair pigmentation and consequent photosensitivity, actinic damage and risk of skin cancer, and by reduced visual acuity and nystagmus. Our survey of Japanese patients revealed that OCA1 was the most frequent type at 34%, while type 2 was present at less than 10%. OCA3 was absent. OCA4, which is a rare type worldwide, was the second most frequent type at 27%. Unexpectedly 10% of the patients turned out to be Hermansky-Pudlak syndrome type 1. Furthermore, the pathogenic p.A481T allele for OCA2, which has 70% melanogenesis activity, was found in approximately 12% of normally pigmented people, indicating that sub-clinical OCA2 might be more frequent in the Japanese than currently thought. And OCA4 is one of the most common types in Japanese patients, despite being rare worldwide.

Background ADAMs (a disintegrin and metalloprotease) are a family of proteases involved in ectodomain shedding that play a role in various biological processes such as cell adhesion and migration. ADAM10 and ADAM17 are suggested to be involved in pigmentary disorders. Objective We examined the effect of ADAM protease inhibitors on the modulation of melanogenesis in normal human epidermal melanocytes (NHEM). Methods NHEMs and B16F10 treated with ADAM protease inhibitors were analyzed. AlamarBlue cell proliferation assay, melanin content assay, tyrosinase activity assay, Western blotting analysis, electron microscopic analysis, and RNA interference were employed. Results In NHEMs, melanin content was reduced by treatment with ADAM protease inhibitors. The inhibitors did not change the protein expression of tyrosinase, TRP-1, and MITF. In B16F10 cells, treatment of the cells with ADAM protease inhibitor diminished the α-MSH-induced increase in melanin content. Electron microscopy showed that the number of fibrillar and mature melanosomes was significantly reduced and that the vacuolar compartments were filled with dense unstructured aggregates after treatment with ADAM protease inhibitors. We therefore focused on the processing of PMEL17, a melanosomal glycoprotein that forms a fibrillar matrix on which melanin gets deposited. Proteolytic processing of PMEL17 is required to form functional fibrils during melanogenesis. Recently, γ-secretase and β-site amyloid precursor protein-cleaving enzyme 2 (BACE2) were found to cleave PMEL17. We found that ADAM protease inhibitors exerted effects on the processing of C-terminal and N-terminal fragments of PMEL17. Using BACE2 siRNA and γ-secretase inhibitor, we showed that ADAM protease inhibitor affected PMEL17 processing in a γ-secretase and BACE2-independent mechanism. Conclusion Several proteases, including ADAM proteases, can contribute to the formation of fibrils and their assembly into sheets in melanosomes.

As reported in the mass media on July 2013, numerous consumers who had used the cosmetic ingredient containing rhododendrol (4-(4-hydroxyphenyl)-2-butanol, Trade name; rhododenol), which is a melanin inhibitor isolated from Acer nikoense Maxim, released from Kanebo Cosmetics Inc. (Tokyo, Japan) noticed leukoderma patches on their face, neck and hands. We have experienced 32 cases that developed leukoderma after using such cosmetics so far and skin biopsy samples in some cases were obtained from both leukoderma and pigmented lesions. A histopathological analysis for skin lesions obtained from such patients notably showed basal hypo-pigmentation, melanin incontinence, and remaining melanocytes in most patients which is not relevant in vitiligo vulgaris. Subsequently, we comprehensively carried out immunohistochemical analyses of immune-competent cells infiltration to assess the effect of the cellular immune response to inducible hypopigmentation. Furthermore, detailed morphological observations performed by electron-microscopy notably showed the presence of melanocytes with only a small number of melanosomes, dermal fibroblasts containing melanosome globules and melanophages whereas no damage associated with melanosome transfer and the basal layer apparatus. These findings provide a cue to diagnose as rhododenol-induced leukoderma differentiate from vitiligo vulgaris and for rhododendrol to induce local immunity in addition to melanocyte damage.