Moesin deficiency leads to lupus-like nephritis with accumulation of CXCL13-producing patrolling monocytes.

Moesin is a member of the ezrin-radixin-moesin (ERM) family of proteins that link plasma membrane proteins to the cortical cytoskeleton and thus regulate diverse cellular processes. Mutations in the human moesin gene cause a primary immunodeficiency called X-linked moesin-associated immunodeficiency (X-MAID), which may be complicated by an autoimmune phenotype with kidney involvement. We previously reported that moesin-deficient mice exhibit lymphopenia similar to that of X-MAID and develop a lupus-like autoimmune phenotype with age. However, the mechanism through which moesin defects cause kidney pathology remains obscure. Here, we characterized immune cell infiltration and chemokine expression in the kidney of moesin-deficient mice. We found accumulation of CD4+ T and CD11b+ myeloid cells and high expression of CXCL13, whose upregulation was detected before the onset of overt nephritis. CD4+ T cell population contained IFN-γ-producing effectors and expressed the CXCL13 receptor CXCR5. Among myeloid cells, Ly6Clo patrolling monocytes and MHCIIlo macrophages markedly accumulated in moesin-deficient kidneys and expressed high CXCL13 levels, implicating the CXCL13-CXCR5 axis in nephritis development. Functionally, Ly6Clo monocytes from moesin-deficient mice showed reduced migration toward sphingosine 1-phosphate. These findings suggest that moesin plays a role in regulating patrolling monocyte homeostasis, and that its defects lead to nephritis associated with accumulation of CXCL13-producing monocytes and macrophages.

Prooxidative effect of cardols is involved in their cytotoxic activity against murine B16-F10 melanoma cells.

Cardols are resorcinolic lipids, available in many natural sources including cashew nut, pistachio, macadamia, and mango. Despite of several beneficial biological activities of cardols, cytotoxic activities of cardols are not fully understood. In preliminary studies, 5[8(Z),11(Z),14-pentadecatrienyl]resorcinol, known as cardol (C15:3) was found to inhibit tyrosinase-catalyzed melanin formation in cell-free system. In the case of cultured cell analysis, cardol (C15:3) showed intense cytotoxicity but not anti-melanogenic activity against B16-F10 melanoma cells. Subsequently, cardol (C10:0) and cardol (C5:0), containing shorter alkyl side chain, exhibited inferior cytotoxicity compared to cardol (C15:3). The cytotoxicity via cardol (C15:3) was reversed by the addition of antioxidants, indicating that intracellular prooxidative activity was involved. Furthermore, cardol (C15:3) produced significant levels of reactive oxygen species (ROS) while cardol (C5:0) generated lesser ROS levels. Our findings suggest the cytotoxic activity of cardols is their prooxidative effect depending on the length of alkyl side chain.

The ERM protein moesin regulates natural killer cell homeostasis in vivo.

Moesin is a member of the ezrin-radixin-moesin (ERM) family of proteins that link plasma membrane proteins with actin filaments in the cell cortex. Hemizygous mutations in the X-linked moesin gene are associated with primary immunodeficiency with T and B cell lymphopenia, which also affects natural killer (NK) cells in most cases. We previously showed that moesin deficiency in mice substantially affects lymphocyte homeostasis, but its impact on NK cells remains unexplored. Here, we found that in moesin-deficient mice, NK cells were decreased in the peripheral blood and bone marrow but increased in the spleen. Analysis of female heterozygous mice showed a selective advantage for moesin-expressing NK cells in the blood. Moesin-deficient NK cells exhibited increased cell death and impaired signaling in response to IL-15, suggesting that moesin regulates NK cell survival through IL-15-mediated signaling. Our findings thus identify moesin as an NK cell homeostasis regulator in vivo.

The Effect of Ethanol on Disassembly of Amyloid-ß1-42 Pentamer Revealed by Atomic Force Microscopy and Gel Electrophoresis.

The most common type of dementia, Alzheimer's disease, is associated with senile plaques formed by the filamentous aggregation of hydrophobic amyloid-ß (Aß) in the brains of patients. Small oligomeric assemblies also occur and drugs and chemical compounds that can interact with such assemblies have attracted much attention. However, these compounds need to be solubilized in appropriate solvents, such as ethanol, which may also destabilize their protein structures. As the impact of ethanol on oligomeric Aß assembly is unknown, we investigated the effect of various concentrations of ethanol (0 to 7.2 M) on Aß pentameric assemblies (Aßp) by combining blue native-PAGE (BN-PAGE) and ambient air atomic force microscopy (AFM). This approach was proven to be very convenient and reliable for the quantitative analysis of Aß assembly. The Gaussian analysis of the height histogram obtained from the AFM images was correlated with band intensity on BN-PAGE for the quantitative estimation of Aßp. Our observations indicated up to 1.4 M (8.3%) of added ethanol can be used as a solvent/vehicle without quantitatively affecting Aß pentamer stability. Higher concentration induced significant destabilization of Aßp and eventually resulted in the complete disassembly of Aßp.

Citrullinated fibrinogen is a target of auto-antibodies in interstitial lung disease in mice with collagen-induced arthritis.

Interstitial lung disease (ILD) is a very common and lethal complication of rheumatoid arthritis (RA), yet its pathogenesis is not well understood, in part due to the lack of adequate animal models. Although collagen-induced arthritis (CIA) is the most widely used animal model for RA, the lung involvement occurring in this model has scarcely been studied. To evaluate the suitability of CIA as a model for RA-associated ILD (RA-ILD), we immunized DBA/1 mice with bovine type II collagen and characterized lung disease in this model. Histologic analyses revealed patchy interstitial infiltration of inflammatory cells in the peripheral regions of the lung, notably in the subpleural region, in mice with CIA. This pattern resembled usual interstitial pneumonia in humans, which is the most prevalent pattern in RA-ILD. Among infiltrates in the lung, CD11bhi macrophages of the M2 phenotype were most prominently increased. IgG and C3 were deposited in the subpleural region where inflammatory cells infiltrated. The sera from CIA mice contained auto-antibodies against citrullinated proteins, which are specific and predictive markers for RA. Protein citrullination was enhanced in the lung of CIA mice compared with naive mice, and citrullinated fibrinogen was primarily targeted by these auto-antibodies. The elevation of auto-antibodies against citrullinated proteins and their deposition in the lung with patchy subpleural preponderance suggest that CIA can serve as a model to study the pathogenesis of RA-ILD.

A novel Siglec-F+ neutrophil subset in the mouse nasal mucosa exhibits an activated phenotype and is increased in an allergic rhinitis model.

Neutrophils are important phagocytic cells for host defense against pathogens. They are rapidly recruited to the site of infection, release antimicrobial peptides and cytokines, and engulf and kill microbes. Neutrophils also accumulate in allergic inflammatory sites. Here we characterized neutrophil accumulation in the nasal mucosa using a mouse model of allergic rhinitis, in which mice were sensitized by intraperitoneal injection of ovalbumin (OVA) and then challenged by intranasal administration of OVA or PBS. In the nasal mucosa of both PBS- and OVA-challenged mice, we found a cell subset expressing the eosinophil marker Siglec-F in the Ly-6G+ neutrophil population. Morphological analysis of the sorted Ly-6G+Siglec-F+ cells revealed that they were devoid of eosinophilic granules in the cytosol and were apparently neutrophils, but compared to conventional Ly-6G+Siglec-F- neutrophils, they had a more lobulated, "botryoid" nucleus. Siglec-F+ neutrophils were barely found in the nasopharynx-associated lymphoid tissue, cervical lymph nodes, the spleen, or blood. Both Siglec-F+ neutrophils and conventional neutrophils showed increased numbers in the nasal mucosa of OVA-challenged mice. Compared to conventional Siglec-F- neutrophils, Siglec-F+ neutrophils exhibited an activated phenotype and enhanced effector functions. Taken together, our findings identify Siglec-F+ neutrophils as a novel neutrophil subset with an activated phenotype that resides specifically in the nasal mucosa.

The ERM Protein Moesin Regulates CD8+ Regulatory T Cell Homeostasis and Self-Tolerance.

The ezrin-radixin-moesin (ERM) proteins are a family of membrane-associated proteins that link membrane proteins with actin filaments in the cell cortex and regulate many cellular processes, including cell shape determination, membrane transport, and signal transduction. Lymphocytes predominantly express two ERM members, ezrin and moesin. Mutations in the moesin gene in humans are associated with primary immunodeficiency with profound lymphopenia, and moesin-deficient mice exhibit a similar lymphopenia phenotype. In this study, we show that aging moesin-deficient mice develop a systemic lupus erythematosus-like autoimmune phenotype, which is characterized by elevated serum autoantibody levels and glomerulonephritis. Younger moesin-deficient mice exhibited elevated basal levels of several Ig isotypes and enhanced Ab affinity maturation upon immunization. Germinal center B cells and follicular helper T cells spontaneously accumulated in unimmunized mice, and CD8+CD44+CD122+Ly49+ regulatory T (CD8+ Tregs) cells, which inhibit the expansion of follicular helper T cells, were severely reduced in these mice. Isolated CD8+ Treg cells from moesin-deficient mice showed impaired proliferation in response to IL-15, which was accompanied by defects in STAT5 activation and IL-15Rα internalization, suggesting that moesin plays a key role in IL-15-mediated signaling. These findings underscore the importance of moesin in IL-15-dependent CD8+ Treg cell homeostasis and, thus, the control of self-tolerance.

Effects of matsutake mushroom scent compounds on tyrosinase and murine B16-F10 melanoma cells.

Tyrosinase-catalyzed l-tyrosine oxidation is a key step in melanogenesis, and intense melanin formation is often a problem in chemotherapies or food preservation. Here we report that methyl cinnamate one of the constituents characterized from mycelium and sporocarp of American matsutake mushroom Tricholoma magnivelare inhibits both enzymatic and cellular melanin formation. Methyl cinnamate inhibits mushroom tyrosinase-catalyzed l-tyrosine oxidation while the oxidation of l-3,4-dihydroxyphenylalanine (l-DOPA) was not inhibited. In subsequent cellular assays, methyl cinnamate significantly suppressed melanogenesis of murine B16-F10 melanoma cells without affecting cell growth. However, methyl 3-phenylpropionate, a dihydro-derivative of methyl cinnamate, did not possess melanogenesis, indicating that the double bond in the enone moiety is a key Michael reaction acceptor to elicit the activity. In addition, a rather rare chlorinated benzaldehyde derivative, 3,5-dichloro-4-methoxybenzaldehyde isolated from the same source, was found to show potent cytotoxicity, and the chlorine atom reduced a tyrosinase inhibitory activity but enhanced cytotoxicity. Our findings suggest that methyl cinnamate is a novel melanogenesis inhibitor from natural sources.

Role of mitochondrial hydrogen peroxide induced by intermittent hypoxia in airway epithelial wound repair in vitro.

The airway epithelium acts as a frontline barrier against various environmental insults and its repair process after airway injury is critical for the lung homeostasis restoration. Recently, the role of intracellular reactive oxygen species (ROS) as transcription-independent damage signaling has been highlighted in the wound repair process. Both conditions of continuous hypoxia and intermittent hypoxia (IH) induce ROS. Although IH is important in clinical settings, the roles of IH-induced ROS in the airway repair process have not been investigated. In this study, we firstly showed that IH induced mitochondrial hydrogen peroxide (H2O2) production and significantly decreased bronchial epithelial cell migration, prevented by catalase treatment in a wound scratch assay. RhoA activity was higher during repair process in the IH condition compared to in the normoxic condition, resulting in the cellular morphological changes shown by immunofluorescence staining: round cells, reduced central stress fiber numbers, pronounced cortical actin filament distributions, and punctate focal adhesions. These phenotypes were replicated by exogenous H2O2 treatment under the normoxic condition. Our findings confirmed the transcription-independent role of IH-induced intracellular ROS in the bronchial epithelial cell repair process and might have significant implications for impaired bronchial epithelial cell regeneration.

Involvement of aquaporin-3 in epidermal growth factor receptor signaling via hydrogen peroxide transport in cancer cells.

Aquaporin 3 (AQP3), a water/glycerol channel protein, is capable of transporting hydrogen peroxide (H2O2). Here, we show that AQP3-mediated intracellular H2O2 is involved in epidermal growth factor (EGF)-induced cell signaling and its dependent cell function in the EGF receptor (EGFR)-positive cancer cell lines A431 and H1666. AQP3 knockdown suppressed the transport into the cells of extracellular H2O2 produced in response to EGF in A431 and H1666 cells. EGF-induced Erk and Akt activation, which occurred through SHP2 and/or PTEN modulation, was impaired by AQP3 knockdown. Cell growth and migration induced by EGF stimulation were attenuated in AQP3 knockdown cells compared with those in control cells. Coincidentally, tumor growth of A431 cell xenografts in immunodeficient mice was decreased by AQP3 knockdown. Accordingly, a xenograft with AQP3 knockdown A431 cells significantly enhanced the survival of recipient mice compared with the transplantation with control cells. In addition, AQP3 associated with EGFR and NADPH oxidase 2, which we propose is linked to AQP3 producing a localized increase in intracellular H2O2 to function as a second messenger during EGFR cell signaling. Therefore, our findings suggest that AQP3 is required for EGF-EGFR cell signaling in cancer cells and is a therapeutic target for cancer progression.

Resveratrol as a kcat type inhibitor for tyrosinase: potentiated melanogenesis inhibitor.

Resveratrol exhibited the inhibitory activity against mushroom tyrosinase (EC1.14.18.1) through a k(cat) inhibition. Resveratrol itself did not inhibit tyrosinase but rather was oxidized by tyrosinase. In the enzymatic assays, resveratrol did not inhibit the diphenolase activity of tyrosinase when l-3,4-dihydroxyphenylalanin (L-DOPA) was used as a substrate; however, L-tyrosine oxidation by tyrosinase was suppressed in presence of 100 µM resveratrol. Oxidation of resveratrol and inhibition of L-tyrosine oxidation suggested the inhibitory effects of metabolites of resveratrol on tyrosinase. After the 30 min of preincubation of tyrosinase and resveratrol, both monophenolase and diphenolase activities of tyrosinase were significantly suppressed. This preincubational effect was reduced with the addition of L-cysteine, which indicated k(cat) inhibition or suicide inhibition of resveratrol. Furthermore, investigation was extended to the cellular experiments by using B16-F10 murine melanoma cells. Cellular melanin production was significantly suppressed by resveratrol without any cytotoxicity up to 200 µM. trans-Pinosylvin, cis-pinosylvin, dihydropinosylvin were also tested for a comparison. These results suggest that possible usage of resveratrol as a tyrosinase inhibitor and a melanogenesis inhibitor.

Isolation of TCR genes with tumor-killing activity from tumor-infiltrating and circulating lymphocytes in a tumor rejection cynomolgus macaque model.

To develop effective adoptive cell transfer therapy using T cell receptor (TCR)-engineered T cells, it is critical to isolate tumor-reactive TCRs that have potent anti-tumor activity. In humans, tumor-infiltrating lymphocytes (TILs) have been reported to contain CD8+PD-1+ T cells that express tumor-reactive TCRs. Characterization of tumor reactivity of TILs from non-human primate tumors could improve anti-tumor activity of TCR-engineered T cells in preclinical research. In this study, we sought to isolate TCR genes from CD8+PD-1+ T cells among TILs in a cynomolgus macaque model of tumor transplantation in which the tumors were infiltrated with CD8+ T cells and were eventually rejected. We analyzed the repertoire of TCRα and ß pairs obtained from single CD8+PD-1+ T cells in TILs and circulating lymphocytes and identified multiple TCR pairs with high frequency, suggesting that T cells expressing these recurrent TCRs were clonally expanded in response to tumor cells. We further showed that the recurrent TCRs exhibited cytotoxic activity to tumor cells in vitro and potent anti-tumor activity in mice transplanted with tumor cells. These results imply that this tumor transplantation macaque model recapitulates key features of human TILs and can serve as a platform toward preclinical studies of non-human primate tumor models.

Alteration in endometrial helper T-cell subgroups in chronic endometritis.

PROBLEM: The effect of chronic endometritis (CE) on the subpopulation of CD4+ T cells, Th1, Th2, Th17, and regulatory T cells in the endometrium is unknown. METHOD OF STUDY: Lymphocytes were isolated from the endometrium of CE patients (n = 12) and non-CE patients (n = 7). The CD4+ T-cell profile was analyzed by flow cytometry and immunofluorescence. RESULTS: In the endometrium of CE patients, there were significantly more Th1 cells among CD4+ cells and fewer Th2 cells in comparison to non-CE patients. No marked difference was observed in Th17 cells or Foxp3+ Treg cells. Moreover, the proportion of Th1 cells increased and the proportion of Th2 cells decreased as the number of CD138+ cells increased. Furthermore, when the localization of CD138+ cells and CD4+ cells was examined, CD4+ cells were found to be clustered around CD138+ cells in CE patients. CONCLUSION: The CD4+ T-cell profile in the endometrium is altered in women with CE. This finding may help to clarify the pathophysiology and development of treatment methods for CE.

Characterization of tumour-infiltrating lymphocytes in a tumour rejection cynomolgus macaque model.

Immunotherapy has emerged as a promising and effective treatment for cancer, yet the clinical benefit is still variable, in part due to insufficient accumulation of immune effector cells in the tumour microenvironment. Better understanding of tumour-infiltrating lymphocytes (TILs) from nonhuman primate tumours could provide insights into improving effector cell accumulation in tumour tissues during immunotherapy. Here, we characterize TILs in a cynomolgus macaque tumour model in which the tumours were infiltrated with CD4+ and CD8+ T cells and were eventually rejected. The majority of CD4+ and CD8+ TILs exhibited a CD45RA-CCR7- effector memory phenotype, but unlike circulating T cells, they expressed CD69, a marker for tissue-resident memory T (TRM) cells. CD69-expressing CD8+ TILs expressed high levels of the cytotoxic molecule granzyme B and the co-inhibitory receptor PD-1. Consistent with the TRM cell phenotype, CD8+ TILs minimally expressed CX3CR1 but expressed CXCR3 at higher levels than circulating CD8+ T cells. Meanwhile, CXCL9, CXCL10 and CXCL11, chemokine ligands for CXCR3, were expressed at high levels in the tumours, thus attracting CXCR3+CD8+ T cells. These results indicate that tumour-transplanted macaques can be a useful preclinical model for studying and optimizing T cell accumulation in tumours for the development of new immunotherapies.

Polygonum odoratum essential oil inhibits the activity of mushroom derived tyrosinase.

Plant derived compounds are a source of long term research focus due to their applications in a variety of fields, particularly food preservation. One key way in which phytochemicals are crucial in this area is by disrupting enzyme functionality. In this work, essential oil was extracted by steam distillation from the fresh leaves of Polygonum odoratum (Polygonaceae), commonly known as Vietnamese coriander, and shown to effectively inhibit the oxidation of L-3,4-dihydroxyphenylalanine (L-DOPA) catalyzed by mushroom tyrosinase (EC1.14.18.1). Using GC-MS analysis, twenty five compounds were identified in the essential oil. The most abundant compounds in the essential oil were Alkanals - dodecanal (55.49%), and decanal (11.57%) - followed by anisaldehyde (6.35%); these compounds were individually investigated for inhibitory activity by performing single-compound screening. Each of the top three most abundant compounds inhibited the tyrosinase-catalyzed oxidation of L-DOPA, as identified by UV-VIS spectroscopy and oxygen consumption assays. The inhibitory activity of the major compounds increased when pre-incubated with tyrosinase and without significant additional oxygen consumption, suggesting k cat -type inactivation is not involved. Interactions of the head and tail components of the major alkanals may disrupt the tertiary structure of the enzyme, presenting a potential inhibitory mechanism.

Aquaporin-3 Controls Breast Cancer Cell Migration by Regulating Hydrogen Peroxide Transport and Its Downstream Cell Signaling.

Most breast cancer mortality is due to clinical relapse associated with metastasis. CXCL12/CXCR4-dependent cell migration is a critical process in breast cancer progression; however, its underlying mechanism remains to be elucidated. Here, we show that the water/glycerol channel protein aquaporin-3 (AQP3) is required for CXCL12/CXCR4-dependent breast cancer cell migration through a mechanism involving its hydrogen peroxide (H2O2) transport function. Extracellular H2O2, produced by CXCL12-activated membrane NADPH oxidase 2 (Nox2), was transported into breast cancer cells via AQP3. Transient H2O2 accumulation was observed around the membrane during CXCL12-induced migration, which may be facilitated by the association of AQP3 with Nox2. Intracellular H2O2 then oxidized PTEN and protein tyrosine phosphatase 1B (PTP1B) followed by activation of the Akt pathway. This contributed to directional cell migration. The expression level of AQP3 in breast cancer cells was related to their migration ability both in vitro and in vivo through CXCL12/CXCR4- or H2O2-dependent pathways. Coincidentally, spontaneous metastasis of orthotopic xenografts to the lung was reduced upon AQP3 knockdown. These findings underscore the importance of AQP3-transported H2O2 in CXCL12/CXCR4-dependent signaling and migration in breast cancer cells and suggest that AQP3 has potential as a therapeutic target for breast cancer.

Aquaporin-3-mediated hydrogen peroxide transport is required for NF-κB signalling in keratinocytes and development of psoriasis.

Aquaporin 3 (AQP3), a water/glycerol channel protein, has been found to transport hydrogen peroxide (H2O2). Here, we show that H2O2, imported via AQP3, is involved in nuclear factor-κB (NF-κB) signalling in keratinocytes and in the pathogenesis of psoriasis. IL-23-mediated induction of psoriasis is reduced in AQP3 knockout mice (AQP3(-/-)), and is accompanied by impaired NF-κB activation and intracellular H2O2 accumulation. In primary keratinocyte cultures, cellular import of H2O2 produced by membrane NADPH oxidase 2 (Nox2) in response to TNF-α is facilitated by AQP3 and required for NF-κB activation by regulation of protein phosphatase 2A. As AQP3 associates with Nox2, we propose that this interplay constitutes H2O2-mediated signalling in response to TNF-α stimulation. Collectively, these data indicate that AQP3-facilitated H2O2 transport is required for NF-κB activation in keratinocytes in the development of psoriasis.

Effects of thymol on B16-F10 melanoma cells.

Aromatic monoterpene, thymol, shows several beneficial activities, such as an antioxidative effect. However, the mechanism of its toxicity remains to be fully defined. In preliminary studies, thymol was characterized as a melanin formation inhibitor in an enzymatic system; however, thymol showed moderate cytotoxicity but not an antimelanogenic effect on B16-F10 melanoma cells. Thymol exhibited cytotoxicity, with an IC(50) value of 400 µM (60.09 µg/mL). This moderate toxic effect was suppressed with the addition of vitamin C and vitamin D, and 20 and 40% of cell viability was increased, respectively. Subsequently, the treatment of L-cysteine on thymol-treated melanoma cells reversed the toxic effect of thymol. Moreover, a significant oxidative stress condition was observed when B16 melanoma cells were cultured with thymol. In conclusion, the antioxidant actions of thymol generate a stable phenoxy radical intermediate, which generates reactive oxygen species and quinone oxide derivatives. Thus, it is proposed that the primary mechanism of thymol toxicity at high doses is due to the formation of antioxidant-related radicals.

Melanogenesis inhibitors from Rabdosia japonica.

The effects of the four major ent-kaurene diterpenoids isolated from the aerial part of Rabdosia japonica (Labiatae) on murine B16-F10 melanoma cells were investigated. Among the compounds tested, oridonin and nodosin most significantly suppressed cellular melanin production when the cells were cultured with these diterpenoids. However, oridonin and nodosin exhibited cytotoxicity against the same melanoma cells with an IC(50) of 1.1 µM (0.40 µg/ml) and of 1.3 µM (0.47 µg/ml) and almost complete lethality was observed at 4.0 µM and at 8.0 µM, respectively, and therefore observed melanogenesis inhibition is mainly due to its melanocytotoxic effect. Morphological observation showed that oridonin or nodosin treated B16-F10 melanoma cells induced dendrite structure. Diterpenoids quickly formed adducts partly in Dulbecco's Modified Eagle's Medium (DMEM) containing 10% of fetal bovine serum (10% FBS-DMEM) before their application to the cells. Approximately 20% of oridonin formed adducts within the first 15 min. Notably, dihydronodosin exhibited inferior cytotoxicity (>85% cell viability at 100 µM) but still significantly suppressed melanogenesis (>55%) when murine B16-F10 melanoma cells were cultured with this diterpenoid derivatives. Hence, dihydronodosin can be a potential melanogenesis inhibitor.

Effects of thymol on mushroom tyrosinase-catalyzed melanin formation.

The novel inhibitory mechanism of thymol (2-isopropyl-5-methylphenol) on dopachrome formation by mushroom tyrosinase (EC 1.14.18.1) was identified. The UV-vis spectrum and oxygen consumption assays showed dopachrome formation using L-tyrosine as a substrate was suppressed by thymol. This inhibitory activity was reversed by the addition of a well-known radical scavenger, butylated hydroxyanisole (BHA). Further investigations using N-acetyl-L-tyrosine as a substrate with HPLC analysis suggested that thymol inhibits chemical redox reactions between dopaquinone and leukodopachrome instead of enzymatic reaction. This redox inhibitory activity of thymol was examined by using a model redox reaction with L-dihydroxyphenylalanine (L-DOPA) and p-benzoquinone. Thymol successfully inhibited oxidation of L-DOPA to dopaquinone, coupled with reduction of p-benzoquinone. Hence, the suppression of dopachrome formation by thymol is due to the inhibition of conversion of leukodopachrome to dopachrome. The antioxidant property of thymol is a key characteristic for the inhibitory mechanism of melanin synthesis.