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The mechanism of melanocytes-specific cytotoxicity induced by phenol compounds having a prooxidant effect, relating to the appearance of leukoderma.

Nagata T, Ito S, Itoga K, Kanazawa H, Masaki H - Biomed Res Int (2015)

Bottom Line: Furthermore, although raspberry ketone (RK), RD derivative, also increased intracellular ROS in B16F10 cells, increase in ROS was suppressed by disodium dihydrogen ethylenediaminetetraacetate dehydrate (EDTA).The amounts of increased ROS with RK in HaCaT cells without melanocyte were further increased by tyrosinase.Therefore, tyrosinase, a metalloprotein having copper, was speculated to be one of causative agents allowing phenol compounds to work as a prooxidant.

View Article: PubMed Central - PubMed

Affiliation: Institute of Advanced Biomedical Engineering and Science, Tokyo Women's Medical University, 2-10 Kawadacho, Shinjuku-ku, Tokyo 162-0054, Japan ; I.T.O. Co. Ltd., 1-6-7-3F Naka-cho, Musashino, Tokyo 180-0006, Japan.

ABSTRACT
Specific phenol compounds including rhododendrol (RD), a skin-brightening ingredient in cosmetics, are reported to induce leukoderma, inducing a social problem, and the elucidation of mechanism of leukoderma is strongly demanded. This study investigated the relationship among the cytotoxicities of six phenol compounds on B16F10 melanoma cells and HaCaT keratinocytes and generated reactive oxygen species (ROS). As a result, the cytotoxicity of RD on B16F10 cells was higher than that on HaCaT cells, and RD significantly increased intracellular ROS and hydrogen peroxide (H2O2) levels in B16F10 cells. Furthermore, although raspberry ketone (RK), RD derivative, also increased intracellular ROS in B16F10 cells, increase in ROS was suppressed by disodium dihydrogen ethylenediaminetetraacetate dehydrate (EDTA). The amounts of increased ROS with RK in HaCaT cells without melanocyte were further increased by tyrosinase. Therefore, tyrosinase, a metalloprotein having copper, was speculated to be one of causative agents allowing phenol compounds to work as a prooxidant. Hydroxyl radical was generated by adding a mixture of tyrosinase and H2O2 to RD, and the amount of the radical was further increased by UVB, indicating that RD cytotoxicity was caused by intracellularly increased ROS, which possibly related to phenol induced prooxidants.

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Related in: MedlinePlus

Relationships among the cell viabilities of (a) B16F10 melanoma cells and (b) HaCaT keratinocytes, which were treated with 5.0 mM rhododendrol (RD), raspberry ketone (RK), hydroquinone monomethyl ether (MEHQ), p-hydroxyphenylacetamide (pHPA), 2-(p-hydroxyphenyl)isovaleric acid (2pHP-IA), or p-hydroxyphenylethanol (pHPE), and the generated amounts of ROS in both cells, which were treated with 1.0 mM of the phenolic compounds. Pearson correlation coefficients (r) were calculated, and the probability less than 0.05 (P < 0.05) was considered statistically significant.
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fig5: Relationships among the cell viabilities of (a) B16F10 melanoma cells and (b) HaCaT keratinocytes, which were treated with 5.0 mM rhododendrol (RD), raspberry ketone (RK), hydroquinone monomethyl ether (MEHQ), p-hydroxyphenylacetamide (pHPA), 2-(p-hydroxyphenyl)isovaleric acid (2pHP-IA), or p-hydroxyphenylethanol (pHPE), and the generated amounts of ROS in both cells, which were treated with 1.0 mM of the phenolic compounds. Pearson correlation coefficients (r) were calculated, and the probability less than 0.05 (P < 0.05) was considered statistically significant.

Mentions: After B16F10 cells were treated with 1.0 mM RK, which is made by replacing the hydroxyl group of RD with ketone group, for 30 min, the generation of ROS was clearly confirmed in the cells (Figure 3). Since the intensity of fluorescence emitted from B16F10 cells treated with 5.0 mM RK was too high to be measured with the instrument for measuring intracellular ROS level, RK concentration was reduced from 5.0 to 1.0 mM and the experiment which used B16F10 cells treated with 0.1 mM RK was performed. Similarly, after B16F10 and HaCaT cells were treated with phenolic compounds including 1.0 mM RD, RK, MEHQ, pHPA, 2pHP-IA, or pHPE for 30 min, the generated amounts of ROS in the cells were determined, and only by the treatments with RD, RK, and MEHQ, the increased amounts of ROS in B16F10 cells were found to be higher than those of HaCaT cells (Figure 4). The correlations between the cell viabilities of B16F10 and HaCaT cells, which were treated with 5.0 mM RD, RK, MEHQ, pHPA, 2pHP-IA, and pHPE, and the generated amounts of ROS in both cells, which were treated with 1.0 mM of the phenolic compounds, were investigated (Figure 5). Although a significant negative correlation was found between the cell viabilities and the generated amounts of ROS in B16F10 cells (Figure 5(a)), no significant correlation was confirmed in HaCaT cells (Figure 5(b)), indicating that only B16F10 cells showed decrease in cell viability with increasing the generated amounts of ROS.


The mechanism of melanocytes-specific cytotoxicity induced by phenol compounds having a prooxidant effect, relating to the appearance of leukoderma.

Nagata T, Ito S, Itoga K, Kanazawa H, Masaki H - Biomed Res Int (2015)

Relationships among the cell viabilities of (a) B16F10 melanoma cells and (b) HaCaT keratinocytes, which were treated with 5.0 mM rhododendrol (RD), raspberry ketone (RK), hydroquinone monomethyl ether (MEHQ), p-hydroxyphenylacetamide (pHPA), 2-(p-hydroxyphenyl)isovaleric acid (2pHP-IA), or p-hydroxyphenylethanol (pHPE), and the generated amounts of ROS in both cells, which were treated with 1.0 mM of the phenolic compounds. Pearson correlation coefficients (r) were calculated, and the probability less than 0.05 (P < 0.05) was considered statistically significant.
© Copyright Policy - open-access
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC4377363&req=5

fig5: Relationships among the cell viabilities of (a) B16F10 melanoma cells and (b) HaCaT keratinocytes, which were treated with 5.0 mM rhododendrol (RD), raspberry ketone (RK), hydroquinone monomethyl ether (MEHQ), p-hydroxyphenylacetamide (pHPA), 2-(p-hydroxyphenyl)isovaleric acid (2pHP-IA), or p-hydroxyphenylethanol (pHPE), and the generated amounts of ROS in both cells, which were treated with 1.0 mM of the phenolic compounds. Pearson correlation coefficients (r) were calculated, and the probability less than 0.05 (P < 0.05) was considered statistically significant.
Mentions: After B16F10 cells were treated with 1.0 mM RK, which is made by replacing the hydroxyl group of RD with ketone group, for 30 min, the generation of ROS was clearly confirmed in the cells (Figure 3). Since the intensity of fluorescence emitted from B16F10 cells treated with 5.0 mM RK was too high to be measured with the instrument for measuring intracellular ROS level, RK concentration was reduced from 5.0 to 1.0 mM and the experiment which used B16F10 cells treated with 0.1 mM RK was performed. Similarly, after B16F10 and HaCaT cells were treated with phenolic compounds including 1.0 mM RD, RK, MEHQ, pHPA, 2pHP-IA, or pHPE for 30 min, the generated amounts of ROS in the cells were determined, and only by the treatments with RD, RK, and MEHQ, the increased amounts of ROS in B16F10 cells were found to be higher than those of HaCaT cells (Figure 4). The correlations between the cell viabilities of B16F10 and HaCaT cells, which were treated with 5.0 mM RD, RK, MEHQ, pHPA, 2pHP-IA, and pHPE, and the generated amounts of ROS in both cells, which were treated with 1.0 mM of the phenolic compounds, were investigated (Figure 5). Although a significant negative correlation was found between the cell viabilities and the generated amounts of ROS in B16F10 cells (Figure 5(a)), no significant correlation was confirmed in HaCaT cells (Figure 5(b)), indicating that only B16F10 cells showed decrease in cell viability with increasing the generated amounts of ROS.

Bottom Line: Furthermore, although raspberry ketone (RK), RD derivative, also increased intracellular ROS in B16F10 cells, increase in ROS was suppressed by disodium dihydrogen ethylenediaminetetraacetate dehydrate (EDTA).The amounts of increased ROS with RK in HaCaT cells without melanocyte were further increased by tyrosinase.Therefore, tyrosinase, a metalloprotein having copper, was speculated to be one of causative agents allowing phenol compounds to work as a prooxidant.

View Article: PubMed Central - PubMed

Affiliation: Institute of Advanced Biomedical Engineering and Science, Tokyo Women's Medical University, 2-10 Kawadacho, Shinjuku-ku, Tokyo 162-0054, Japan ; I.T.O. Co. Ltd., 1-6-7-3F Naka-cho, Musashino, Tokyo 180-0006, Japan.

ABSTRACT
Specific phenol compounds including rhododendrol (RD), a skin-brightening ingredient in cosmetics, are reported to induce leukoderma, inducing a social problem, and the elucidation of mechanism of leukoderma is strongly demanded. This study investigated the relationship among the cytotoxicities of six phenol compounds on B16F10 melanoma cells and HaCaT keratinocytes and generated reactive oxygen species (ROS). As a result, the cytotoxicity of RD on B16F10 cells was higher than that on HaCaT cells, and RD significantly increased intracellular ROS and hydrogen peroxide (H2O2) levels in B16F10 cells. Furthermore, although raspberry ketone (RK), RD derivative, also increased intracellular ROS in B16F10 cells, increase in ROS was suppressed by disodium dihydrogen ethylenediaminetetraacetate dehydrate (EDTA). The amounts of increased ROS with RK in HaCaT cells without melanocyte were further increased by tyrosinase. Therefore, tyrosinase, a metalloprotein having copper, was speculated to be one of causative agents allowing phenol compounds to work as a prooxidant. Hydroxyl radical was generated by adding a mixture of tyrosinase and H2O2 to RD, and the amount of the radical was further increased by UVB, indicating that RD cytotoxicity was caused by intracellularly increased ROS, which possibly related to phenol induced prooxidants.

Show MeSH
Related in: MedlinePlus