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Background: The tyrosinase inhibitor rhododendrol (RD), used as a skin whitening agent, reportedly has the potential to induce leukoderma., Objective: Although an immune response toward melanocytes was demonstrated to be involved in leukoderma, the molecular mechanism is not fully understood., Methods: We hypothesized that if RD is a pro-hapten and tyrosinase-oxidized RD metabolites are melanocyte-specific sensitizers, the sensitizing process could be reproduced by the human cell line activation test (h-CLAT) cocultured with melanocytes (h-CLATw/M) composed of human DC THP-1 cells and melanoma SK-MEL-37 cells. Cell surface expression, ROS generation and ATP release, mRNA expression, and the effects of several inhibitors were examined., Results: When RD was added to the h-CLATw/M, the expression of cell-surface CD86 and IL-12 mRNA was greatly enhanced in THP-1 cells compared with those in the h-CLAT. The rapid death of melanoma cells was induced, with ROS generation and ATP release subsequently being greatly enhanced, resulting in the cooperative upregulation of CD86 and IL-12. Consistent with those observations, an ROS inhibitor, ATP receptor P2X7 antagonist, or PERK inhibitor antagonized the upregulation. CD86 upregulation was similarly observed with another leukoderma-inducible tyrosinase inhibitor, raspberry ketone, but not with the leukoderma noninducible skin-whitening agents ascorbic acid and tranexamic acid., Conclusion: RD is a pro-hapten sensitizer dependent on tyrosinase that induces ROS generation and ATP release from melanocytes for CD86 and IL-12 upregulation in DCs, possibly leading to the generation of tyrosinase-specific cytotoxic T lymphocytes. The coculture system h-CLATw/M may be useful for predicting the sensitizing potential to induce leukoderma., Competing Interests: Declaration of Competing Interest Kazuyuki Yo, Fumiya Yamaji, and Akemi Toyoda are employees of POLA Chemical Industries, Inc., (Copyright © 2022 The Author(s). Published by Elsevier B.V. All rights reserved.)

Idiopathic leukoderma is a skin disorder characterized by patchy loss of skin pigmentation due to melanocyte dysfunction or deficiency. Rhododendrol (RD) was approved as a cosmetic ingredient in Japan in 2008. However, it was shown to induce leukoderma in approximately 20,000 customers. The prediction of cytotoxicity, especially to melanocytes in vivo, is required to avoid such adverse effects. Since the use of higher vertebrates is prohibited for medicinal and toxicological assays, we used zebrafish, whose melanocytes were regulated by mechanisms similar to mammals. Zebrafish larvae were treated with RD in breeding water for 3 days, which caused body lightening accompanied by a decrease in the number of melanophores. Interestingly, black particles were found at the bottom of culture dishes, suggesting that the melanophores peeled off from the body. In addition, RT-PCR analysis suggested that the mRNA levels of melanophore-specific genes were significantly low. An increase in the production of reactive oxygen species was found in larvae treated with RD. The treatments of the fish with other phenol compounds, which have been reported to cause leukoderma, also induced depigmentation and melanophore loss. These results suggest that zebrafish larvae could be used for the evaluation of leukoderma caused by chemicals, including RD., (© 2021 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

Background: Rhododendrol (4-(4-hydroxyphenyl)-2-butanol) has been used as a lightening/whitening cosmetic but was recently reported to induce leukoderma. Although rhododendrol has been shown to be transformed by tyrosinase to hydroxyl-rhododendrol, which is cytotoxic to melanocytes, its detailed mechanism of action including the involvement of reactive oxygen species is not clearly understood., Objective: To confirm the relationship of hydroxyl radical generation to melanocyte cytotoxicity induced by rhododendrol, this study was performed., Methods: An electron spin resonance method with a highly sensitive detection system was utilized to monitor hydroxyl radicals generated from two distinct normal human epidermal melanocyte lines with different levels of tyrosinase activity after the addition of various amounts of rhododendrol. Cytotoxicity of rhododendrol was analyzed by AlamarBlue assay under the same condition., Results: Hydroxyl radicals were generated depending on the amounts of rhododendrol and/or tyrosinase. After the correlation between hydroxyl radical generation with melanocyte viability was confirmed, an inhibitor of oxidative stress, N-acetyl cysteine, was shown to dramatically diminish rhododendrol-induced generation of hydroxyl radicals and melanocyte cytotoxicity by increasing glutathione levels. In contrast, buthionine sulfoximine, which depletes glutathione, augmented both of those parameters., Conclusion: Suppressing oxidative stress would prevent and/or mitigate some phenol derivative-induced leukoderma by avoiding hydroxyl radical-initiated melanocyte cytotoxicity., (Copyright © 2018 The Authors. Published by Elsevier B.V. All rights reserved.)

Rhododendrol is a phenolic compound that shows a tyrosinase-dependent toxicity for melanocytes and occasionally induces a vitiligo-like skin depigmentation. The post-tyrosinase mechanisms determining melanocyte death or survival, however, are far from clear. Here, we find that rhododendrol treatment leads to a reduction in the levels of cellular glutathione but also induces a cellular antioxidant response that eventually increases glutathione levels. We further find that rhododendrol toxicity is enhanced when glutathione levels are experimentally reduced and alleviated when glutathione levels are increased. Hence, it appears that the size of the preexisting glutathione pool along with the capacity to supply glutathione via the antioxidant response determines whether melanocytes survive or die after rhododendrol exposure. It is conceivable, therefore, that rhododendrol-induced leukoderma depends on the capacity to maintain appropriate glutathione levels and that enhancement of glutathione levels may preserve a patient's melanocytes and potentially help in repigmentation., (© 2016 The Authors. Pigment Cell & Melanoma Research published by John Wiley & Sons Ltd.)

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., (Copyright © 2014 Japanese Society for Investigative Dermatology. Published by Elsevier Ireland Ltd. All rights reserved.)