Original articles| Volume 108, ISSUE 2, P77-86, November 2022

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A cell-based evaluation of human tyrosinase-mediated metabolic activation of leukoderma-inducing phenolic compounds

Published:December 12, 2022DOI:


      • The tyrosinase-mediated formation of o-quinone from phenols can trigger leukoderma.
      • Ectopic expression of human tyrosinase converts rhododendrol to quinone metabolites.
      • Rhododendrol-quinones generated bind to glutathione and cysteine in this cell model.
      • A series of leukoderma-inducing phenols is converted to quinone-thiol adducts.
      • Thiol-adduct analysis is more sensitive and specific than cytotoxicity assays.



      Chemical leukoderma is a skin depigmentation disorder induced through contact with certain chemicals, most of which have a p-substituted phenol structure similar to the melanin precursor tyrosine. The tyrosinase-catalyzed oxidation of phenols to highly reactive o-quinone metabolites is a critical step in inducing leukoderma through the production of melanocyte-specific damage and immunological responses.


      Our aim was to find an effective method to evaluate the formation of o-quinone by human tyrosinase and subsequent cellular reactions.


      Human tyrosinase-expressing 293T cells were exposed to various phenolic compounds, after which the reactive o-quinones generated were identified as adducts of cellular thiols. We further examined whether the o-quinone formation induces reductions in cellular GSH or viability.


      Among the chemicals tested, all 7 leukoderma-inducing phenols/catechol (rhododendrol, raspberry ketone, monobenzone, 4-tert-butylphenol, 4-tert-butylcatechol, 4-S-cysteaminylphenol and p-cresol) were oxidized to o-quinone metabolites and were detected as adducts of cellular glutathione and cysteine, leading to cellular glutathione reduction, whereas 2-S-cysteaminylphenol and 4-n-butylresorcinol were not. In vitro analysis using a soluble variant of human tyrosinase revealed a similar substrate-specificity. Some leukoderma-inducing phenols exhibited tyrosinase-dependent cytotoxicity in this cell model and in B16BL6 melanoma cells where tyrosinase expression was effectively modulated by siRNA knockdown.


      We developed a cell-based metabolite analytical method to detect human tyrosinase-catalyzed formation of o-quinone from phenolic compounds by analyzing their thiol-adducts. The detailed analysis of each metabolite was superior in sensitivity and specificity compared to cytotoxicity assays for detecting known leukoderma-inducing phenols, providing an effective strategy for safety evaluation of chemicals.


      4SCAP (4-S-cysteaminylphenol), 5SCD (5-S-cysteinyldopa), 5SGD (5-S-glutathionyldopa), BTQ (dihydro-1,4-benzothiazine-6,7-dione), CRE (p-cresol), Cys (cysteine), Cys-BOC (Cys-benzyloxycatechol), Cys-MeC (Cys-methylcatechol), Cys-RDC (Cys-RD-catechol), Cys-RKC (Cys-RK-catechol), EM (eumelanin), ER (endoplasmic reticulum), GS- (glutathionyl-), GS-BOC (GS-benzyloxycatechol), GSH (glutathione), GS-RDC (GS-RD-catechol), GS-RKC (GS-RK-catechol), GS-MeC (GS-methylcatechol), GS-TBC (GS-TB-catechol), MBEH (monobenzyl ether of hydroquinone), NAC (N- acetylcysteine), PM (pheomelanin), RD (rhododendrol, RDC, RD-catechol), RK (raspberry ketone), ROS (reactive oxygen species), RUC (rucinol), TBP (4-tert-butylphenol), TBC (4-tert-butylcatechol)


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