research article the mechanism of melanocytes-specific...

Research ArticleThe Mechanism of Melanocytes-Specific CytotoxicityInduced by Phenol Compounds Having a Prooxidant Effectrelating to the Appearance of Leukoderma

Takeshi Nagata12 Shinobu Ito23 Kazuyoshi Itoga1

Hideko Kanazawa3 and Hitoshi Masaki4

1 Institute of Advanced Biomedical Engineering and Science Tokyo Womenrsquos Medical University 2-10 KawadachoShinjuku-ku Tokyo 162-0054 Japan2ITO Co Ltd 1-6-7-3F Naka-cho Musashino Tokyo 180-0006 Japan3Faculty of Pharmacy Keio University 1-5-30 Shibakoen Minato-ku Tokyo 105-8512 Japan4School of Bioscience and Biotechnology Tokyo University of Technology 1404-1 Katakuramachi Hachioji Tokyo 192-0982 Japan

Correspondence should be addressed to Kazuyoshi Itoga itogakazuyoshitwmuacjp

Received 25 August 2014 Accepted 1 March 2015

Academic Editor Davinder Parsad

Copyright copy 2015 Takeshi Nagata et alThis is an open access article distributed under the Creative Commons Attribution Licensewhich permits unrestricted use distribution and reproduction in any medium provided the original work is properly cited

Specific phenol compounds including rhododendrol (RD) a skin-brightening ingredient in cosmetics are reported to induceleukoderma inducing a social problem and the elucidation of mechanism of leukoderma is strongly demanded This studyinvestigated the relationship among the cytotoxicities of six phenol compounds on B16F10melanoma cells andHaCaT keratinocytesand generated reactive oxygen species (ROS) As a result the cytotoxicity of RD on B16F10 cells was higher than that on HaCaTcells and RD significantly increased intracellular ROS and hydrogen peroxide (H

2O2) levels in B16F10 cells Furthermore although

raspberry ketone (RK) RD derivative also increased intracellular ROS in B16F10 cells increase in ROSwas suppressed by disodiumdihydrogen ethylenediaminetetraacetate dehydrate (EDTA) The amounts of increased ROS with RK in HaCaT cells withoutmelanocyte were further increased by tyrosinase Therefore tyrosinase a metalloprotein having copper was speculated to be oneof causative agents allowing phenol compounds to work as a prooxidant Hydroxyl radical was generated by adding a mixture oftyrosinase andH

2O2to RD and the amount of the radical was further increased byUVB indicating that RD cytotoxicity was caused

by intracellularly increased ROS which possibly related to phenol induced prooxidants

1 Introduction

In 2013 leukoderma has been reported to appear on theskin of women who use particular skin-brightening cosmeticproducts in Japan The damage has been found widely notonly in Japan but also in Asia inducing a major socialproblem More than 5000 people have leukoderma in Japanat August 2013 Since skin-brightening cosmetic productscontaining a specific phenol compound called rhododen-drol (4-(4-hydroxyphenyl)-2-butanol (RD)) (Figure 1(a)) arereported to induce leukoderma the elucidation of possi-ble mechanism of leukoderma induced by RD is stronglydemanded Although leukoderma is reported to be inducedby the death of melanocytes [1] RD is still unknown to showa specific cytotoxicity to melanocytes

Melanocytes produce melanin actively as their character-istic features and the cells have an indispensable enzymetyrosinase [2] Tyrosinase is known to have copper in itsactive center [3ndash5] Further proteins containing copper areknown to enhance oxidation reactions and induce apoptosis[6 7] Moreover a metal chelating agent such as disodiumdihydrogen ethylenediaminetetraacetate dehydrate (EDTA)inhibits the oxidation reaction of transition metals such asiron and copper with hydrogen peroxide (H

2O2) which

induces Fenton reaction [8 9]On the other hand phenolic compounds including RD

are used as building blocks for preparing various pharmaceu-tical compounds cosmetic ingredients and food additivesbecause of their special characters in oxidation-reduction

Hindawi Publishing CorporationBioMed Research InternationalVolume 2015 Article ID 479798 12 pageshttpdxdoiorg1011552015479798

2 BioMed Research International

HO

OH

(a)

HO

O

(b)

O

HO

(c)

HO

O

NH2

(d)

HOO

OH

(e)

HO

OH

(f)

HO

O

OH

NH2

(g)

Figure 1 Chemical structures of phenolic compounds (a) Rhododendrol (RD) (b) raspberry ketone (RK) (c) hydroquinone monomethylether (MEHQ) (d) p-hydroxyphenylacetamide (pHPA) (e) 2-(p-hydroxyphenyl)isovaleric acid (2pHP-IA) (f) p-hydroxyphenylethanol(pHPE) and (g) L-tyrosine

(redox) reactions However some phenolic compounds areknown to show specific cytotoxicities to melanocytes Forinstance raspberry ketone (4-(4-hydroxyphenyl)-2-butan-one (RK)) (Figure 1(b)) which is made by replacing the hy-droxyl group of RD with ketone group hydroquinone (HQ)hydroquinone monomethyl ether (MEHQ) (Figure 1(c))4-tertiary-butylphenol (4-TBP) 2-phenylphenol and p-octylphenol have been known to cause leukoderma [10ndash16]

There are a few studies that directly detect the generationof reactive oxygen species (ROS) generated bymetalloproteinin vitro Actually the authors have already confirmed thatoxidation reactions promoted with iron proteins such ashemoglobin enhance the toxicity in ultraviolet- (UV-) ray-irradiation induced inflamed cutaneous tissues by comparinggenerated ROS quantitatively with electron spin resonance(ESR) spectroscopy [17]

Recently the cytotoxicity of RD in melanocyte has beenreported to be suppressed by phenylthiourea a copperchelating agent and the knockdown of tyrosinase [18] Thisreport indicates that the cytotoxicity of RD in melanocytesappears through the intermediary compounds of tyrosinaseFurthermore the cytotoxicity of RD in melanocytes has beenfound to be involved in the oxidation of RDby tyrosinase [19]For elucidating the cytotoxicity mechanism in melanocytesby RD difference in the cytotoxicities between cells with andwithout melanin should be investigated

This study therefore investigated the relationshipsamong generated ROS and the cytotoxicities of RD inB16F10 melanoma cells and HaCaT keratinocytes havingno melanin and compared the results to those of RKMEHQ p-hydroxyphenylacetamide (pHPA) (Figure 1(d))2-(p-hydroxyphenyl)isovaleric acid (2pHP-IA) (Figure 1(e))and p-hydroxyphenylethanol (pHPE) (Figure 1(f)) Thisstudy also investigated whether RD could induce the genera-tion of H

2O2 which is known to induce directly the Fenton

reaction in B16F10 cells Hydroxyl radical (OH∙) was directlymeasured by ESR for confirming that the Fenton reactionwasinduced by H

2O2and copper with tyrosinase Furthermore

the oxidation pathway of RD to catechol derivatives by

tyrosinase was also investigated by high performance liquidchromatography (HPLC) analysis

2 Methods

21 Materials RK pHPA 2pHP-IA pHPE and H2O2were

purchased from Wako Pure Chemical Industries (OsakaJapan) RD and EDTA were purchased from Nacalai Tesque(Kyoto Japan) MEHQ was purchased from Tokyo Chem-ical Industry (Tokyo Japan) respectively Tyrosinase frommushroom neutral red and Triton X-100 reduced werepurchased from Sigma-Aldrich (St Louis MO USA) and2101584071015840-dichlorofluorescin diacetate (DCFDA) was purchasedfrom EMD Millipore (Billerica MA) Hoechst33342 waspurchased from Life Technologies (Carlsbad CA) 55-Dimethyl-1-pyrroline-N-oxide (DMPO) was purchased fromLabotec (Tokyo) Dimethyl sulfoxide (DMSO) was pur-chased from Pierce Biotechnology (Rockford IL) Ferroussulfate was purchased from United States PharmacopeialConvention (Rockville MD) Dulbeccorsquos modified Eaglemedium (DMEM) was purchased from Nissui Pharmaceu-tical (Tokyo) Fetal bovine serum (FBS) was purchased fromBiological Industries Israel (Kibbutz Beit-Haemek Israel)

22 Cell Culture B16F10 cells were cultured in DMEMcontaining 10 FBS at 37∘C at a 5-CO

2condition HaCaT

cells were cultured in DMEM containing 5 FBS at 37∘C at a5-CO

2condition

23 Cell Viability B16F10 and HaCaT cells were cultured inDMEM containing 50mM RD RK MEHQ pHPA 2pHP-IA pHPE or 100mM MEHQ and pHPA individually andfor measuring the survival rates the medium was replacedwithDMEMcontaining 0165mgmLneutral red After beingcultured for 2 h the cells were washed with phosphatebuffered saline (PBS) and neutral red incorporated in thecells was extracted with NR extraction solution which wasa mixture of methanol acetic acid and water at a ratioof 50 1 49 The absorbance of neutral red in a well was

BioMed Research International 3

measured at 550 nm with a reference absorbance of 650 nmby a microplate reader (VMax Kinetic ELISA MicroplateReader) (Molecular Devices Sunnyvale CA) The cell sur-vival rates were expressed as percent values of those of thecells incubated without test compounds described above

24 Intracellular ROS Level B16F10 cells were cultured inHanksrsquo balanced salt solution (HBSS) containing 10mMRK200120583MDCFDA and 40 120583MHoechst33342 for 30minAfterbeing incubated for 30min the cells were washed with HBSSand observed with a fluorescence microscope (EVOS FLoidCell Imaging Station) (Life Technologies Carlsbad CA)

B16F10 and HaCaT cells were cultured in HBSS con-taining 10mM RD RK MEHQ pHPA 2pHP-IA or pHPEand 200120583M DCFDA for 30min washed with HBSS andsolubilized with 05 Triton X-100 reducedThe fluorescenceintensity of the medium containing the solubilized cells wasmeasured at an exciting wavelength of 485 nm and an emis-sion wavelength of 530 nm with a fluorescence microplatereader (Gemini EM fluorescence microplate reader) (Molec-ular Devices) The intracellular ROS level was expressedas a relative value which was calculated from fluorescenceintensity per 10 120583g protein of the control cells incubatedwithout test samples

The effects of tyrosinase and EDTA on change in intra-cellular ROS by exposure to RK were investigated by thefollowing procedure HaCaT cells were cultured in HBSScontaining 500 750 or 1000 unitsmL tyrosinase with10mM RK and 200 120583M DCFDA for 30min B16F10 cellswere cultured in HBSS containing 025 05 or 10mM EDTAwith 10mM RK and 200120583M DCFDA for 30min Thefluorescence intensity of the medium was measured by thesame method described above The intracellular ROS levelwas expressed as a relative value which was calculated fromfluorescence intensity per 10120583g protein of the control cellsincubated without tyrosinase EDTA and RK

25 Intracellular H2O2 Level After being cultured in HBSScontaining 50mM RD or RK for 30min B16F10 cells werewashed with HBSS and solubilized with 05 Triton X-100reduced and the intracellular amount of H

2O2wasmeasured

with Amplite fluorimetric hydrogen peroxide assay kit ldquoRedFluorescencerdquo (ATT Bioquest Sunnyvale CA) The amountof H2O2was expressed as a relative value which was calcu-

lated fromfluorescence intensity per 10120583g protein of the con-trol cells incubated without test samples such as RD and RK

26 ESR Signal Measurement A JES-FA200 ESR spectrom-etry (JEOL Tokyo) was used for ESR signal measurementESR spectrometry conditions used for estimating radicalspecies with adequate spin-trap reagents were as followsmicrowave frequency 9414499 plusmn 5000MHz microwavepower 400mW field center 33532 plusmn 05mT sweep widthplusmn500mT modulation frequency 10000 kHz sweep time05sim5min amplitude 1500sim2500 and time constant 003sim05 s at room temperature This study used an ESR univer-sal cavity (ES-UCX2 TE011-mode cavity) (JEOL) with X-bandmicrowave units (8750ndash9650GHz) an aqueous samplecell (ES-LC12 JEOL) with a sample volume of 20sim100120583L

and a quartz cell (Labotec) with a home-made cover glass(40 times 5 times 05mm) As an ESR standard marker manganeseoxide (MnO) powder (MO7-FB-4 JEOL) was used As aspin-trap agent 55-dimethyl-1-pyrroline-N-oxide (DMPO100ww liquid) and 10ww DMSO solution were usedThe following test samples were solved in 01M PBS (pH 75)RD (100 120583M) was added to 100 unitsmL tyrosinase DMPO(20120583L) was added to the sample solutions (20120583L) immedi-ately and the radical signals were measured by ESR UVB(290sim340 nm) irradiation was introduced to samples at adose of 200sim1000 Jm2 by a USHIO Optical Modulex (UshioTokyo) To identify observed peaks signals were analyzed byspecialized ESR analysis software (A-System vl40 ISAJ FA-manager vl20) (JES Tokyo) which was installed in the ESRdevice and allowed to determine 119892-value and calculated hfccfrom the distance between peaks To identify ESR signalsthe 119892-value and hfcc of the measurable peaks were comparedwith the peak values of the standard OH∙ The standardOH∙ was generated by the reaction of ferrous sulfate solution(100 nM) and H

2O2(30mM)

27 HPLC Analysis After 1000 120583M RD was added to1000 unitsmL tyrosinase the sample mixture was incubatedfor 0 15 and 30min at 37∘C and was injected into an HPLCinstrument Sample (500 120583L) was separated on an octade-cylsilane (ODS) reverse-phase column (Shodex C18P-4Ecolumn) (Showa Denko Tokyo) at 40∘C The mobile phasewhich was a mixture of acetonitrile and 003M KH

2PO4at a

ratio of 60 40 was used at a flow rate of 07mLmin TheHPLC system consisted of a SCL-10A system controller aLC-10AD pump a SPD-10A UVVIS detector a CTO-10ACcolumn oven and a SIL-10A injector (Shimadzu Kyoto)Theseparated peaks were monitored at 300 nm with the detector

28 Protein Assay Protein concentration was determinedwith bicinchoninic acid- (BCA-) protein assay kit (PierceBiotechnology)

29 Statistical Analysis Comparison between two groupswas performed by Studentrsquos t-test and the probability lessthan 005 (119875 lt 005) was considered statistically significantCorrelations were analyzed by calculating Pearson correla-tion coefficients (119903)

3 Results

31 The Cytotoxicities of Phenol Compounds to B16F10 andHaCaT Cells Figure 2 shows the survival rates of B16F10 andHaCaT cells treated with RD RK MEHQ pHPA 2pHP-IAor pHPE for 24 h The survival rates of HaCaT cells treatedwith 50mMRDandRKwere found to be significantly higherthan those of B16F10 cells The survival rates of HaCaT cellstreated with 100mM MEHQ were found to be significantlyhigher than those of B16F10 cells The results indicated thatRD RK and MEHQ had a specific cytotoxicity to B16F10cells However the survival rates of B16F10 cells treated with50mM 2pHP-IA pHPE and 100mM pHPA were found tobe significantly higher than those of HaCaT cells indicating


0

20

40

60

80

100

120

RD RK

MEH

Q

pHPA

2pH

P-IA

pHPE

MEH

Q

pHPA

Surv

ival

rate

()

HaCaTB16F10

50 mM 100mM

lowastlowastlowastlowast


lowastlowast

lowastlowast

Figure 2 Effects of phenol compounds on the survival rates of B16F10melanoma cells andHaCaT keratinocytes B16F10 andHaCaT cells weretreated with 50mM rhododendrol (RD) raspberry ketone (RK) hydroquinone monomethyl ether (MEHQ) p-hydroxyphenylacetamide(pHPA) 2-(p-hydroxyphenyl)isovaleric acid (2pHP-IA) p-hydroxyphenylethanol (pHPE) 100mMMEHQ or pHPA for 24 h Cell survivalrates were determined by using neutral red The black and gray columns indicate the averaged survival rates of B16F10 and HaCaT cellsrespectively The lines on the columns show the standard deviations (119899 = 5) Two asterisks (lowastlowast) indicate that the probabilities of significancelevels are less than 001 (119875 lt 001)

that 2pHP-IA pHPE and pHPA showed no B16F10 cells-specific cytotoxicity

32 Effects of Phenol Compounds on the Intracellular ROSLevels of B16F10 and HaCaT Cells After B16F10 cells weretreated with 10mM RK which is made by replacing thehydroxyl group of RDwith ketone group for 30min the gen-eration of ROS was clearly confirmed in the cells (Figure 3)Since the intensity of fluorescence emitted from B16F10 cellstreated with 50mM RK was too high to be measured withthe instrument for measuring intracellular ROS level RKconcentration was reduced from 50 to 10mM and theexperiment which used B16F10 cells treated with 01mM RKwas performed Similarly after B16F10 and HaCaT cells weretreated with phenolic compounds including 10mM RD RKMEHQ pHPA 2pHP-IA or pHPE for 30min the generatedamounts of ROS in the cells were determined and onlyby the treatments with RD RK and MEHQ the increasedamounts of ROS in B16F10 cells were found to be higher thanthose of HaCaT cells (Figure 4) The correlations betweenthe cell viabilities of B16F10 and HaCaT cells which weretreated with 50mM RD RK MEHQ pHPA 2pHP-IA andpHPE and the generated amounts of ROS in both cells whichwere treated with 10mM of the phenolic compounds wereinvestigated (Figure 5) Although a significant negative corre-lationwas found between the cell viabilities and the generatedamounts of ROS in B16F10 cells (Figure 5(a)) no significantcorrelation was confirmed in HaCaT cells (Figure 5(b))indicating that only B16F10 cells showed decrease in cellviability with increasing the generated amounts of ROS

33 Effect of Tyrosinase on Intracellular ROS Induced by RKTyrosinase was investigated to be involved in the increase

in intracellular ROS level As a result 500 750 and1000 unitsmL tyrosinase were confirmed to further promoteincrease in the intracellular ROS level in HaCaT cells treatedwith 10mM RK (Figure 6)

34 Effect of EDTA on Intracellular ROS Induced by RKEDTA a metal chelating agent was investigated to decreaseROS generated in B16F10 cells with RK intracellularly As aresult 05 and 10mM EDTA were confirmed to reduce theintracellular ROS level increased with 10mM RK (Figure 7)

35 Effect of RD on the Intracellular H2O2 of B16F10 CellsB16F10 cells were treated with RD and RK and the intra-cellular H

2O2level in the cells was measured As a result

50mM RD and RK were found to increase the H2O2level

significantly (Figure 8)

36 ESR Experiment After RD was added to a sample mix-ture of tyrosinase and H

2O2and irradiated by UVB at a dose

of 1000 Jm2 DPMO-radical adduct spectra of the sampleswere detected by ESR (Figure 9) The heights of ESR peakswhich are marked with downward arrow heads (998787) werefound to increase with RD (Figure 9(c)) and further increasebyUVB irradiation (Figure 9(d))These four waveformswerefound to be derived from hydroxyl radical (OH∙) by their119892-values and hfcc values in the measurable peaks in ESRFigure 10 indicates the relative values of the peak intensitiesin the secondary waves of OH∙ spectra The samples wereirradiated with UVB at 200 500 and 1000 Jm2 and the peakintensity of OH∙ spectra of a sample mixture of tyrosinaseH2O2 and RD was significantly increased at a dose of

1000 Jm2 Further the peak intensity of OH∙ spectra wasfound to increase with UVB irradiation dose-dependently


37 HPLC Experiment After 1000 120583M RD was added to1000 unitsmL tyrosinase the sample mixture was incubatedfor 15 or 30min at 37∘C and was analyzed by HPLC Theseparated peaks were monitored at 310 nm Although thesample gave a peak derived from RD at a retention time (RT)of 58mm (Figure 11(a)) a 15-minute incubation also gaveunknown peaks at 47 50 55 and 64min (Figure 11(b)) Onthe contrary a 30-minute incubation gave only an unknownpeak at 55min which was found to be different from theRD peak (Figure 11(c)) Since a mixture of acetonitrile and003M KH

2PO4at a ratio of 60 40 was used as a mobile

phase in the reverse-phase HPLC column for analyzing thesamples observed RT was expected to be slower for higherhydrophobic substancesTherefore unknown peaks found atearlier than 58min were thought to be higher water-solublesubstances than RD Also an unknown peak at 64minwhichwas obtained fromRDbybeing reactedwith tyrosinasefor 15min (Figure 11(b)) was found to be identical with RKpeak (data not shown)

4 Discussion

RK andMEHQare reported to show their cytotoxicitiesmorestrongly in B16 cells than human fibrosarcoma HT1080 cells[20]However there is no study investigating themelanocyte-specific cytotoxicities of other phenolic compounds includingRD pHPA 2pHP-IA and pHPE This study evaluated themelanocyte-specific cytotoxicities of phenolic compounds byinvestigating the effects of RD RK MEHQ pHPA 2pHP-IAand pHPE on the survival rates against B16F10 and HaCaTcells As a result RD RK and MEHQ which are alreadyreported to cause leukoderma on the skin were found toreduce the survival rate of B16F10 cells more strongly thanthat of HaCaT cells and the B16F10 cells-specific cytotoxici-ties of these phenolic compounds were confirmed (Figure 2)indicating that the cause of leukoderma in the skin due toRD was speculated to be a melanocytes-specific cytotoxicitywhich was similar to those of RK and MEHQ

The productions of ROS inside melanocytes could possi-bly contribute to the appearance of the melanocytes-specificcytotoxicity because normalmelanocytes treatedwith 4-TBPwhich shows a melanocytes-specific cytotoxicity for 30minare reported to increase the intracellular amounts of ROS[21] Moreover the amounts of ROS in the melanocytesof the patients with leukoderma on the skin are reportedto be higher than those of normal melanocytes of healthypeople [22] Naturally this study investigated changes in theintracellular amounts of ROS in B16F10 and HaCaT cellsthat were treated with RD RK MEHQ pHPA 2pHP-IA orpHPE and observed that the amounts of ROS in B16F10 cellstreated with RD RK andMEHQ which are already reportedto give leukoderma on the skin were increased significantlycompared to those ofHaCaT cells (Figure 4) Remarkably RKwas found to increase the intracellular amount of ROS morein B16F10 than HaCaT cells by 1574 These results showedthat the B16F10 cells-specific cytotoxicities of phenolic com-pounds including RD RK and MEHQ were speculated tobe originated from the increased amounts of ROS in B16F10cells

All phenolic compounds used in this study were exam-ined for their cytotoxicities and ROS productions in B16F10and HaCaT cells (Figures 2 and 4) and only RD RK andMEHQ showed a higher cytotoxicity and a higher ROSgenerating ability in B16F10 thanHaCaT cells suggesting thatother phenolic compounds were unnecessary to be testedin further experiments including that with tyrosinase Inspite of their comparable B16F10 cell-specific cytotoxicitiesand ROS generating abilities (Figures 2 and 4) the chemicalstructure of RK is similar to that of RD an approvedcosmetic ingredient but that ofMEHQ is different from them(Figure 1) Therefore only RD and RK were used in furtherexperiments

Melanocytes including B16F10 cells are known to producemelanin for various physiological purposes and the produc-tion process is calledmelanogenesis For performingmelano-genesis actively tyrosinase is known as an indispensableenzyme and has copper in its active center [3ndash5] Transitionmetals including iron and copper are known to react withredox molecules with generating ROS [23] Since phenoliccompounds including RD have a chemical structure similarto that of L-tyrosine (Figure 1(g)) it may act as a substratefor tyrosinase Actually 4-TBP binds to the active center ofthe enzyme and works as a competitive inhibitor [24] Byconsidering the structures of these phenolic compounds thisstudy assumed that the distance between the terminal oxygenatom of the side chain and the benzene ring of phenoliccompounds was important The distances in RD and RKhaving a B16F10 cells-specific cytotoxicity are speculated tobe approximately 05 nm and this distance was assumed tobe substantially the same as that of L-tyrosine This distancewas speculated to be related to the depth of the pocketof the active center in tyrosinase Therefore the phenoliccompounds having the same distance as that of L-tyrosinecould be easily oxidized by tyrosinase

The reaction of RD or RK with copper in tyrosinase hasa possibility to induce the enhancement of oxidation whichcan generate reactive oxygen species (ROS) resulting in thepossible appearance of cytotoxicity eventually by the proox-idant effects of RD and RK Since (1) 500sim1000 unitsmLtyrosinase further increased the intracellular amount of ROSwhich had been increased by 10mM RK in HaCaT cellsby 1442sim2625 significantly (Figure 6) and (2) 05 and10mM EDTA a chelating agent significantly suppressed theintracellular amount of ROS which had been increased by10mM RK in B16F10 cells by approximately 223 and 422respectively (Figure 7) copper in tyrosinase was speculatedto be involved in generating ROSTherefore copper in tyrosi-nase might contribute to the increase in the amount of ROSgenerated by RD in B16F10 cells Reaction of transitionmetalssuch as iron and copper withH

2O2is reported to induce Fen-

ton reaction which is known as a reaction generating highlyand fatally cytotoxic ROS such asOH∙ [25ndash34] In the authorsrsquoprevious study iron proteins such as hemoglobin in the cuta-neous tissue and ascorbic acid (AA) are reported to promoteFenton reaction which enhances UV-irradiation inducedcytotoxicity [17] Reaction of copper and AA is reported topromote Fenton reaction [23] Since the intracellular amountof H2O2in B16F10 cells was significantly increased by RD by


Hoechst33342 DCFDA Merged

Con

trol B

16F1

0B1

6F1

0+10

mM

RK

Figure 3 The fluorescence microphotographs of intracellular reactive oxygen species (ROS) level in B16F10 melanoma cells treated withraspberry ketone (RK) After being treated with 10mMRK 200 120583M2101584071015840-dichlorofluorescin diacetate (DCFDA) and 40 120583MHoechst33342for 30min B16F10 cells were observed with a fluorescencemicroscope DCFDA andHoechst33342 were able to showROS level and the nucleiof the cells respectively Scale bar 100 120583m

00

10

20

30

RD RK MEHQ pHPA 2pHP-IA pHPE

Relat

ive i

ntra

cellu

lar R

OS

leve

l

HaCaTB16F10

lowastlowast

lowastlowast

lowastlowast

01mM

Figure 4 Effects of phenol compounds on the intracellular reactive oxygen species (ROS) level of B16F10 melanoma cells and HaCaT ker-atinocytes After B16F10 and HaCaT cells were treated with 10mM rhododendrol (RD) raspberry ketone (RK) hydroquinone monomethylether (MEHQ) p-hydroxyphenylacetamide (pHPA) 2-(p-hydroxyphenyl)isovaleric acid (2pHP-IA) or p-hydroxyphenylethanol (pHPE)and 200 120583M 2101584071015840-dichlorofluorescin diacetate (DCFDA) for 30min the fluoresce intensities were measured The black and gray columnsindicate the averaged intracellular ROS levels in B16F10 and HaCaT cells respectivelyThe lines on the columns show the standard deviations(119899 = 5) One (lowast) and two (lowastlowast) asterisks indicate that the probabilities of significance levels are less than 005 (119875 lt 005) and 001 (119875 lt 001)respectively

approximately 110 (Figure 8) RD also had a possibility toinduce Fenton reaction with copper in tyrosinase As shownin Figures 9 and 10 OH∙ was found to be generated by thereaction of RD and tyrosinase and OH∙ might be a cause ofthe B16F10 cells-specific cytotoxicity of RD In Figure 9(c)specific 4 peaks derived from OH∙ were confirmed by ESRFurthermore OH∙ generated by the reaction of RD andtyrosinase was found to be increased by UVB irradiationdose-dependently (Figure 10) and the increase agreed with

the fact that RD-induced leukoderma appears more stronglyon UV-exposed sites such as face neck and hands

The substrates of tyrosinase are L-tyrosine L-dopa and56-dihydroxyindole (DHI) [2] L-tyrosine is oxidized toL-dopa a catechol derivative by tyrosinase and L-dopais also oxidized to dopaquinone a quinone derivative bytyrosinase Having a structure similar to that of L-tyrosine4-TBP is reported to be able to bind with the active centerof tyrosinase and work as an inhibitor [24] Having a


00

05

10

15

20

25

30

35

40 60 80 100

Relat

ive i

ntra

cellu

lar R

OS

leve

l

Survival rate ()

y = minus00351x + 43578

r = minus0888

P = 0018

(a)

00

05

10

15

20

60 70 80 90 100

Relat

ive i

ntra

cellu

lar R

OS

leve

l

Survival rate ()

y = minus00064x + 05358

r = 0377

P = 0461

(b)

Figure 5 Relationships among the cell viabilities of (a) B16F10 melanoma cells and (b) HaCaT keratinocytes which were treated with50mM 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 whichwere treated with 10mM of the phenolic compounds Pearson correlation coefficients (119903) were calculated and the probability less than 005(119875 lt 005) was considered statistically significant

00

10

20

30

40

50

60

70

0 500 750 1000

Relat

ive i

ntra

cellu

lar R

OS

leve

l

Tyrosinase (unitsmL)

0mM RK10mM RK

lowastlowast

lowastlowast


Figure 6 Effect of tyrosinase on intracellular reactive oxygen species (ROS) induced by raspberry ketone (RK) After HaCaT keratinocyteswere treated with 500sim1000 unitsmL tyrosinase 10mM RK and 200 120583M 2101584071015840-dichlorofluorescin diacetate (DCFDA) for 30min thefluoresce intensities were measured The black and gray columns indicate the averaged intracellular ROS levels in B16F10 treated with 0 and10mM RK respectively The lines on the columns show the standard deviations (119899 = 5) Two asterisks (lowastlowast) indicate that the probabilities ofsignificant levels are less than 001 (119875 lt 001)

structure more similar to that of L-tyrosine than that of4-TBP RD was speculated to bind with the active centerof tyrosinase Therefore RD as well as L-tyrosine wasspeculated to be oxidized to catechol and quinone derivativesby tyrosinase Actually 4-TBP and 4-tertiary-butyl cate-chol (4-TBC) are reported to be converted into quinone

derivatives by tyrosinase [35] HPLC analysis showed thattwo substances at 47 and 50min which were slower thanRD peak at 58min were found to be generated by thereaction of RD and tyrosinase for 15min (Figure 11(b))As the property of reverse-phase HPLC column RT is knownto be inversely proportional to the number of hydroxyl


0mM RK10mM RK

lowastlowastlowastlowast lowastlowast

00

10

20

30

40

50

0 025 050 100

Relat

ive i

ntra

cellu

lar R

OS

leve

l

EDTA (mM)

Figure 7 Effect of disodium dihydrogen ethylenediaminetetraacetate dihydrate (EDTA) on intracellular reactive oxygen species (ROS)induced by raspberry ketone (RK) After B16F10 melanoma cells were treated with 025sim10mM EDTA 10mM RK and 200 120583M 2101584071015840-dichlorofluorescin diacetate (DCFDA) for 30min the fluoresce intensities were measuredThe black and gray columns indicate the averagedintracellular ROS levels in B16F10 treated with 0 and 10mMRK respectivelyThe lines on the columns show the standard deviations (119899 = 5)Two asterisks (lowastlowast) indicate that the probabilities of significant levels are less than 001 (119875 lt 001)

00

05

10

15

20

Ctl RD RK

Rela

tive i

ntra

cellu

lar H

2O2

leve

l

lowast lowast

50mM

Figure 8 Effect of rhododendrol (RD) on the intracellular hydrogen peroxide (H2O2) level of B16F10melanoma cells After B16F10 cells were

treated with 50mMRD and raspberry ketone (RK) for 15min the intracellular H2O2level of the cells was measured by Amplite fluorimetric

hydrogen peroxide assay kit ldquoRed Fluorescencerdquo The black columns indicate the averaged intracellular H2O2levels in B16F10 treated with

50mM RD and RK The lines on the columns show the standard deviations (119899 = 5) One (lowast) and two (lowastlowast) asterisks indicate that theprobabilities of significance levels are less than 005 (119875 lt 005) and 001 (119875 lt 001) respectively

groups Therefore two substances which appeared at 47and 50min which were made from RD by reacting withtyrosinase for 15min were speculated to havemore hydroxylgroups than RD A substance at 64min was also found to begenerated by the reaction of RD and tyrosinase for 15min

(Figure 11(b)) and might be derived from RK because the RTof this peak agreed with that of RK Since RD is made byadding a hydrogen atom to RK structure RK was speculatedto be generated by the oxidization of RD by tyrosinaseFurthermore when RD reacted with tyrosinase for 30min a


(a) H2O2

(b) H2O2 + tyrosinase

(c) H2O2 + tyrosinase + RD

(d) H2O2 + tyrosinase + RD + 1000 Jm2 UVB

Figure 9 Electron spin resonance (ESR) spectra of 55-dimethyl-1-pyrroline-N-oxide (DMPO) adduct signals in a sample mixtureof hydrogen peroxide (H

2O2) tyrosinase and rhododendrol (RD)

irradiated with ultraviolet B (UVB) at a dose of 1000 Jm2 After100120583M RD was added to 100 unitsmL tyrosinase DMPO (20 120583L)was added to the sample mixture (20 120583L) which was irradiated withUVB immediately and the radical signals weremeasured by ESR (a)H2O2 (b) a mixture of H

2O2and tyrosinase (c) a mixture of H

2O2

tyrosinase and RD and (d) a mixture of H2O2 tyrosinase and RD

and the mixture was irradiated with UVB

substance at 55min was found to be generated (Figure 11(c))This substance was speculated to have fewer hydroxyl groupsthan two substances made from RD by reacting with tyrosi-nase for 15min These results suggested that RD might beconverted into a substance at 55min through two interme-diated substances appearing at 47 and 50min

Figure 12 shows a possible pathway where RD waseventually converted into a quinone derivative throughcatechol and semiquinone derivatives by tyrosinase Acatechol derivative of RD (RD-catechol Figure 12(b))two semiquinone derivatives of RD (RD-semiquinone)(Figures 12(c) and 12(c1015840)) which are prooxidant moleculesof RD and a quinone derivative of RD (RD-quinone)(Figure 12(d)) were arranged in the descending orderof the number of hydroxyl groups RD-catechol gt RD-semiquinone gt RD-quinone When these three substanceswere assumed to be three substances appearing at 47 50 and55min (Figures 11(b) and 11(c)) the results of reverse-phaseHPLC analysis (Figure 11) were able to support a possiblepathway of oxidization of RD quite logically (Figure 12)

Because the oxidation of RD to its quinone derivatives wasspeculated to be performed more possibly by intracellulartyrosinase than extracellular autoxidation the oxidationprocess could closely relate to the intracellular production ofROS in B16F10 melanoma cells This speculation could agreewith the experimental results that the increased intracellularamount of ROS in B16F10 cells having tyrosinase was higherthan that of HaCaT cells having no tyrosinase (Figure 4)

The production of superoxide is enhanced by addingH2O2and dopa to tyrosinase solution [36] Moreover H

2O2

is reported to be generated in oxidization process fromphenol derivative to quinone derivative by copper [37]23-Dihydroxybenzoic acid which is a natural polyphenolwhose structure is similar to that of RD-catechol enhancesoxidation reaction with copper in the presence of H

2O2[38]

H2O2is now well-known to be produced in the generating

process of quinone by the oxidization of phenolic hydroxylgroup of anthrahydroquinone on industrial scale processwhich is called anthraquinone oxidation (AO) process [39]The results of these reports agree with the speculation thatH2O2was generated in the oxidization process of RD in this

study (Figure 12) and RD-quinone might be converted intoRD-quinone radical (RD-quinone∙) through the conjugationof allylic C-H bonds with the 120587-bond (120587-allylic conjugation)[40 41] Therefore since divalent metal oxidizes to univalentmetal with the oxidization of metalloprotein [23 28ndash30] Cu(II) in tyrosinase was assumed to be oxidized to Cu(I) withgenerating hydrogen through the generating pathway of RD-quinone∙ as indicated in the following

Cu(II)-tyrosinase + RD

997888rarr Cu(I)-tyrosinase + RD-quinone∙ +H+(1)

Since the reaction of univalent metalloprotein with H2O2

induces the generation of OH∙ [23 28ndash30] copper intyrosinase was thought to be converted as indicated in thefollowing

Cu(I)-tyrosinase +H2O2

997888rarr Cu(II)-tyrosinase +OH∙ +OHminus(2)

The formation of a 120587-allylic conjugation in phenolic com-pounds suggested that the conjugated system extends to theside chain of phenolic compounds and electrons were ableto move between the terminal oxygen atom of the side chainand the benzene ring of phenolic compounds The distancebetween the terminal oxygen atom of the side chain and thebenzene ring of RD or RK was speculated to be comparableto that of L-tyrosine indicating that the moving distance ofelectrons in RDor RKmight be the same as that in L-tyrosinerelating to amelanocytes-specific cytotoxicity induced by RDor RK

RD is approved to be used as a skin-brightening ingre-dient in quasidrug category in Japan Therefore many safetyevaluation tests for RD have been carried out and thesafety of RD is ensured at the same safety level as that ofmedicine However RD has induced a strong adverse effectleukoderma in 2013 There is a question why RD has passed


00

20

40

60

80

100

Relat

ive E

SR p

eak

inte

nsity

val

ue

RD

200 500 1000

H2O2 H2O2 + tyrosinase

UVB (Jm2)

lowastlowast lowastlowast lowastlowast lowastlowast lowastlowast

lowastlowast

Figure 10 Relative electron spin resonance (ESR) peak intensity values of 55-dimethyl-1-pyrroline-N-oxide (DMPO) adduct signals in asample mixture of hydrogen peroxide (H

2O2) tyrosinase and rhododendrol (RD) and the mixture was irradiated with ultraviolet B (UVB)

After 100 120583M RD was added to 100 unitsmL tyrosinase DMPO (20 120583L) was added to the sample mixture (20 120583L) which was irradiatedwith UVB (200sim1000 Jm2) immediately and the radical signals were measured by ESR The black columns indicate the averaged ESR peakintensityThe lines on the columns show the standard deviations (119899 = 8) Two asterisks (lowastlowast) indicate that the probabilities of significant levelsare less than 001 (119875 lt 001)

58

0

50

100

150

200

Peak

inte

nsity

(mAU

)

40 60 80Retention time (min)

20

(a) 0min

58

55 64

0

50

100

Peak

inte

nsity

(mAU

)

4750


20

(b) 15min

55

40 60

50

100

Peak

inte

nsity

(mAU

)

80Retention time (min)

0

20

(c) 30min

Figure 11 High performance liquid chromatography (HPLC) analysis of rhododendrol (RD) After 1000 120583MRDwas added to 1000 unitsmLtyrosinase the samplemixture was incubated for (a) 0 (b) 15 and (c) 30min at 37∘C and injected into the reverse-phase HPLCThe separatedpeaks were monitored at 310 nm


HO HO O

OHO HO

HO

O

OH OH

OH

OH

OH

Tyrosinase

(a) (b)

(c)

(d)

Tyrosinase+

H2O2

O2

Tyrosinase+O2

∙

∙

O

(c998400)OH∙

Figure 12 A proposed reactive oxygen species (ROS) generation mechanism in B16F10 melanoma cells by rhododendrol (RD) (a) RD (b)RD-catechol (c) RD-semiquinone and (d) RD-quinone

many safety tests This study confirmed that RD induced amelanocytes-specific cytotoxicity and increased an intracel-lular ROS level in B16F10 cells Although three-dimensionalcultured skin models are used in safety evaluation tests thereare no safety evaluation tests using melanocytes Thereforefor improving the safety of quasidrug or cosmetic ingredientsafety evaluation tests which investigate whether a testcompound shows a melanocytes-specific cytotoxicity andincreases an intracellular ROS level in melanocytes aresuggested to be necessary The authors hope that this studyrsquosresults could contribute to the establishments of these safetyevaluation tests

5 Conclusions

RD increased the intracellular H2O2level in B16F10 cells

Since the structure of RD is similar to that of L-tyrosineRD is thought to react with copper in tyrosinase Moreoverthe results of reverse-phase HPLC analysis suggested thatRD was oxidized to a quinone derivative through catecholand semiquinone derivatives by tyrosinase Therefore H

2O2

was speculated to be generated in the oxidization process toquinone derivative from RD in B16F10 cells FurthermoreESR spectroscopy in this study confirmed that OH∙ whichis known to show a high cytotoxicity was generated by thereaction of tyrosinase and H

2O2 indicating that reaction of

copper in tyrosinase andH2O2might induce Fenton reaction

simultaneously This study proposed that the RD-inducedmelanocytes-specific cytotoxicity could be caused possibly byOH∙ generated by the reaction of copper in tyrosinase andH2O2 and this reaction might be one of the mechanisms for

explaining RD-induced leukoderma

Conflict of Interests

The authors declare that they have no conflict of interests inthis paper

Acknowledgments

The authors are grateful to Nobuyuki Nagato and Yui YoshiiITO Co Ltd Tokyo Japan for their encouragement andsupport

References

[1] R E Boissy and P Manga ldquoOn the etiology of contactoccupational vitiligordquo Pigment Cell Research vol 17 no 3 pp208ndash214 2004

[2] A Slominski D J Tobin S Shibahara and J WortsmanldquoMelanin pigmentation in mammalian skin and its hormonalregulationrdquo Physiological Reviews vol 84 no 4 pp 1155ndash12282004

[3] W T Ismaya H J Rozeboom AWeijn et al ldquoCrystal structureof agaricus bisporus mushroom tyrosinase identity of thetetramer subunits and interaction with tropolonerdquo Biochem-istry vol 50 no 24 pp 5477ndash5486 2011

[4] Y Matoba T Kumagai A Yamamoto H Yoshitsu and MSugiyama ldquoCrystallographic evidence that the dinuclear coppercenter of tyrosinase is flexible during catalysisrdquo The Journal ofBiological Chemistry vol 281 no 13 pp 8981ndash8990 2006

[5] C Olivares and F Solano ldquoNew insights into the active sitestructure and catalytic mechanism of tyrosinase and its relatedproteinsrdquo Pigment Cell ampMelanoma Research vol 22 no 6 pp750ndash760 2009

[6] M-C de Pauw-Gillet B Siwek G Pozzi E Sabbioni and R JB Bassleer ldquoControl of B16 melanoma cells differentiation andproliferation by CuSO4 and vitamin Crdquo Anticancer Researchvol 10 no 2 pp 391ndash395 1990

[7] R Stoewe and W A Prutz ldquoCopper-catalyzed dna damageby ascorbate and hydrogen peroxide kinetics and yieldrdquo FreeRadical Biology and Medicine vol 3 no 2 pp 97ndash105 1987

[8] B L Hevroni A H Sayer E Blum and B Fischer ldquoNucleoside-21015840310158403101584051015840- bis(thio)phosphate analogues are promising antiox-idants acting mainly via Cu+Fe2+ ion chelationrdquo InorganicChemistry vol 53 no 3 pp 1594ndash1605 2014

[9] I Spasojevic M Mojovic Z Stevic et al ldquoBioavailability andcatalytic properties of copper and iron for Fenton chemistry inhuman cerebrospinal fluidrdquo Redox Report vol 15 no 1 pp 29ndash35 2010

[10] K A Arndt and T B Fitzpatrick ldquoTopical use of hydroquinoneas a depigmenting agentrdquo Journal of the American MedicalAssociation vol 194 no 9 pp 965ndash967 1965

[11] E Frenk and P Loi-Zedda ldquoOccupational depigmentationdue to a hydroquinone-containing photographic developerrdquoContact Dermatitis vol 6 no 3 pp 238ndash239 1980

[12] Y Fukuda M Nagano andM Futatsuka ldquoOccupational leuko-derma in workers engaged in 4-(p-hydroxyphenyl)-2-butanonemanufacturingrdquo Journal of Occupational Health vol 40 no 2pp 118ndash122 1998


[13] O James R W Mayes and C J Stevenson ldquoOccupationalvitiligo induced by p-tert-butylphenol a systemic diseaserdquoTheLancet vol 310 no 8050 pp 1217ndash1219 1977

[14] P Kersey and C J Stevenson ldquoVitiligo and occupationalexposure to hydroquinone from servicing self-photographingmachinesrdquo Contact Dermatitis vol 7 no 5 pp 285ndash287 1981

[15] N Nishitani and I Hara ldquoCases of leucomelanodermatosiscaused by phenylphenolrdquo Sangyo Igaku vol 13 no 3 pp 218ndash219 1971

[16] J J OrsquoSullivan and C J Stevenson ldquoScreening for occupationalvitiligo in workers exposed to hydroquinonemonomethyl etherand to paratertiary-amyl-phenolrdquo British Journal of IndustrialMedicine vol 38 no 4 pp 381ndash383 1981

[17] S Ito K Itoga M Yamato H Akamatsu and T Okano ldquoTheco-application effects of fullerene and ascorbic acid on UV-Birradiated mouse skinrdquo Toxicology vol 267 no 1ndash3 pp 27ndash382010

[18] M Sasaki M Kondo K Sato et al ldquoRhododendrol a depig-mentation-inducing phenolic compound exerts melanocytecytotoxicity via a tyrosinase-dependent mechanismrdquo PigmentCell amp Melanoma Research vol 27 no 5 pp 754ndash763 2014

[19] S Ito M Ojika T Yamashita and K Wakamatsu ldquoTyrosinase-catalyzed oxidation of rhododendrol produces 2-methylchro-mane-67-dione the putative ultimate toxic metabolite impli-cations for melanocyte toxicityrdquo Pigment Cell amp MelanomaResearch vol 27 no 5 pp 744ndash753 2014

[20] Y Fukuda M Nagano K Tsukamoto and M Futatsuka ldquoInvitro studies on the depigmenting activity of 4-(p-hydroxyphen-yl)-2-butanonerdquo Journal of Occupational Health vol 40 no 2pp 137ndash142 1998

[21] P Manga D Sheyn F Yang R Sarangarajan and R E BoissyldquoA role for tyrosinase-related protein 1 in 4-tert-butylphenol-induced toxicity in melanocytes implications for vitiligordquo TheAmerican Journal of Pathology vol 169 no 5 pp 1652ndash16622006

[22] M L DellrsquoAnna M Ottaviani V Albanesi et al ldquoMembranelipid alterations as a possible basis for melanocyte degenerationin vitiligordquo Journal of Investigative Dermatology vol 127 no 5pp 1226ndash1233 2007

[23] J M C Gutteridge and S Wilkins ldquoCopper-dependenthydroxyl radical damage to ascorbic acid formation of athiobarbituric acid-reactive productrdquo FEBS Letters vol 137 no2 pp 327ndash330 1982

[24] F Yang and R E Boissy ldquoEffects of 4-tertiary butylphenol onthe tyrosinase activity in human melanocytesrdquo Pigment CellResearch vol 12 no 4 pp 237ndash245 1999

[25] A Aroun J L Zhong R M Tyrrell and C Pourzand ldquoIronoxidative stress and the example of solar ultraviolet a radiationrdquoPhotochemical andPhotobiological Sciences vol 11 no 1 pp 118ndash134 2012

[26] P Biemond A J G Swaak H G van Eijk and J F KosterldquoSuperoxide dependent iron release from ferritin in inflamma-tory diseasesrdquo Free Radical Biology and Medicine vol 4 no 3pp 185ndash198 1988

[27] M J Borda A R Elsetinow M A Schoonen and D RStrongin ldquoPyrite-induced hydrogen peroxide formation as adriving force in the evolution of photosynthetic organisms onan early earthrdquo Astrobiology vol 1 no 3 pp 283ndash288 2001

[28] C A Cohn M J Borda and M A Schoonen ldquoRNA decom-position by pyrite-induced radicals and possible role of lipidsduring the emergence of liferdquo Earth and Planetary ScienceLetters vol 225 no 3-4 pp 271ndash278 2004

[29] C A Cohn A Pak D Strongin and M A SchoonenldquoQuantifying hydrogen peroxide in iron-containing solutionsusing leuco crystal violetrdquo Geochemical Transactions vol 6 no3 pp 47ndash51 2005

[30] C A Cohn R Laffers and M A A Schoonen ldquoUsing yeastRNA as a probe for generation of hydroxyl radicals by earthmaterialsrdquo Environmental Science and Technology vol 40 no8 pp 2838ndash2843 2006

[31] J P Famaey ldquoFree radicals and activated oxygenrdquo EuropeanJournal of Rheumatology and Inflammation vol 5 no 4 pp350ndash359 1982

[32] M Hermes-Lima P Ponka and H M Schulman ldquoThe ironchelator pyridoxal isonicotinoyl hydrazone (PIH) and its ana-logues prevent damage to 2-deoxyribose mediated by ferriciron plus ascorbaterdquo Biochimica et Biophysica ActamdashGeneralSubjects vol 1523 no 2-3 pp 154ndash160 2000

[33] A Q Maurıcio G K B Lopes C S Gomes R G Oliveira AAlonso and M Hermes-Lima ldquoPyridoxal isonicotinoyl hydra-zone inhibits iron-induced ascorbate oxidation and ascorbylradical formationrdquo Biochimica et Biophysica Acta vol 1620 no1ndash3 pp 15ndash24 2003

[34] A Menditto D Pietraforte and M Minetti ldquoAscorbic acid inhuman seminal plasma is protected from iron-mediated oxi-dation but is potentially exposed to copper-induced damagerdquoHuman Reproduction vol 12 no 8 pp 1699ndash1705 1997

[35] KThorneby-Andersson O Sterner and C Hansson ldquoTyrosin-asemediated formation of a reactive quinone from the depig-menting agents 4-tert-butylphenol and 4-tert-butylcatecholrdquoPigment Cell Research vol 13 no 1 pp 33ndash38 2000

[36] S Koga M Nakano and S Tero-Kubota ldquoGeneration ofsuperoxide during the enzymatic action of tyrosinaserdquo Archivesof Biochemistry and Biophysics vol 292 no 2 pp 570ndash575 1992

[37] J Y Lee R L Peterson K Ohkubo et al ldquoMechanistic insightsinto the oxidation of substituted phenols via hydrogen atomabstraction by a cupricndashsuperoxo complexrdquo Journal of theAmericanChemical Society vol 136 no 28 pp 9925ndash9937 2014

[38] R Liu B Goodell J Jellison and A Amirbahman ldquoElectro-chemical study of 23-dihydroxybenzoic acid and its interactionwith Cu(II) and H

2O2in aqueous solutions implications for

wood decayrdquo Environmental Science amp Technology vol 39 no1 pp 175ndash180 2005

[39] J M Campos-Martin G Blanco-Brieva and J L G FierroldquoHydrogen peroxide synthesis an outlook beyond the anthra-quinone processrdquo Angewandte ChemiemdashInternational Editionvol 45 no 42 pp 6962ndash6984 2006

[40] K Uwai K Ohashi Y Takaya et al ldquoExploring the structuralbasis of neurotoxicity in C17-polyacetylenes isolated fromwaterhemlockrdquo Journal of Medicinal Chemistry vol 43 no 23 pp4508ndash4515 2000

[41] C Lescot B Darses F Collet P Retailleau and P DaubanldquoIntermolecular C-H amination of complex molecules insightsinto the factors governing the selectivityrdquoThe Journal of OrganicChemistry vol 77 no 17 pp 7232ndash7240 2012

Submit your manuscripts athttpwwwhindawicom

PainResearch and TreatmentHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom

Volume 2014

ToxinsJournal of

VaccinesJournal of

Hindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

AntibioticsInternational Journal of

ToxicologyJournal of


StrokeResearch and TreatmentHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Drug DeliveryJournal of



Advances in Pharmacological Sciences

Tropical MedicineJournal of


Medicinal ChemistryInternational Journal of


AddictionJournal of



BioMed Research International

Emergency Medicine InternationalHindawi Publishing Corporationhttpwwwhindawicom Volume 2014


Autoimmune Diseases


Anesthesiology Research and Practice

ScientificaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of


Pharmaceutics


MEDIATORSINFLAMMATION

of


HO

OH

(a)

HO

O

(b)

O

HO

(c)

HO

O

NH2

(d)

HOO

OH

(e)

HO

OH

(f)

HO

O

OH

NH2

(g)

Figure 1 Chemical structures of phenolic compounds (a) Rhododendrol (RD) (b) raspberry ketone (RK) (c) hydroquinone monomethylether (MEHQ) (d) p-hydroxyphenylacetamide (pHPA) (e) 2-(p-hydroxyphenyl)isovaleric acid (2pHP-IA) (f) p-hydroxyphenylethanol(pHPE) and (g) L-tyrosine

(redox) reactions However some phenolic compounds areknown to show specific cytotoxicities to melanocytes Forinstance raspberry ketone (4-(4-hydroxyphenyl)-2-butan-one (RK)) (Figure 1(b)) which is made by replacing the hy-droxyl group of RD with ketone group hydroquinone (HQ)hydroquinone monomethyl ether (MEHQ) (Figure 1(c))4-tertiary-butylphenol (4-TBP) 2-phenylphenol and p-octylphenol have been known to cause leukoderma [10ndash16]

There are a few studies that directly detect the generationof reactive oxygen species (ROS) generated bymetalloproteinin vitro Actually the authors have already confirmed thatoxidation reactions promoted with iron proteins such ashemoglobin enhance the toxicity in ultraviolet- (UV-) ray-irradiation induced inflamed cutaneous tissues by comparinggenerated ROS quantitatively with electron spin resonance(ESR) spectroscopy [17]

Recently the cytotoxicity of RD in melanocyte has beenreported to be suppressed by phenylthiourea a copperchelating agent and the knockdown of tyrosinase [18] Thisreport indicates that the cytotoxicity of RD in melanocytesappears through the intermediary compounds of tyrosinaseFurthermore the cytotoxicity of RD in melanocytes has beenfound to be involved in the oxidation of RDby tyrosinase [19]For elucidating the cytotoxicity mechanism in melanocytesby RD difference in the cytotoxicities between cells with andwithout melanin should be investigated

This study therefore investigated the relationshipsamong generated ROS and the cytotoxicities of RD inB16F10 melanoma cells and HaCaT keratinocytes havingno melanin and compared the results to those of RKMEHQ p-hydroxyphenylacetamide (pHPA) (Figure 1(d))2-(p-hydroxyphenyl)isovaleric acid (2pHP-IA) (Figure 1(e))and p-hydroxyphenylethanol (pHPE) (Figure 1(f)) Thisstudy also investigated whether RD could induce the genera-tion of H

2O2 which is known to induce directly the Fenton

reaction in B16F10 cells Hydroxyl radical (OH∙) was directlymeasured by ESR for confirming that the Fenton reactionwasinduced by H

2O2and copper with tyrosinase Furthermore

the oxidation pathway of RD to catechol derivatives by

tyrosinase was also investigated by high performance liquidchromatography (HPLC) analysis

2 Methods

21 Materials RK pHPA 2pHP-IA pHPE and H2O2were

purchased from Wako Pure Chemical Industries (OsakaJapan) RD and EDTA were purchased from Nacalai Tesque(Kyoto Japan) MEHQ was purchased from Tokyo Chem-ical Industry (Tokyo Japan) respectively Tyrosinase frommushroom neutral red and Triton X-100 reduced werepurchased from Sigma-Aldrich (St Louis MO USA) and2101584071015840-dichlorofluorescin diacetate (DCFDA) was purchasedfrom EMD Millipore (Billerica MA) Hoechst33342 waspurchased from Life Technologies (Carlsbad CA) 55-Dimethyl-1-pyrroline-N-oxide (DMPO) was purchased fromLabotec (Tokyo) Dimethyl sulfoxide (DMSO) was pur-chased from Pierce Biotechnology (Rockford IL) Ferroussulfate was purchased from United States PharmacopeialConvention (Rockville MD) Dulbeccorsquos modified Eaglemedium (DMEM) was purchased from Nissui Pharmaceu-tical (Tokyo) Fetal bovine serum (FBS) was purchased fromBiological Industries Israel (Kibbutz Beit-Haemek Israel)

22 Cell Culture B16F10 cells were cultured in DMEMcontaining 10 FBS at 37∘C at a 5-CO

2condition HaCaT

cells were cultured in DMEM containing 5 FBS at 37∘C at a5-CO

2condition

23 Cell Viability B16F10 and HaCaT cells were cultured inDMEM containing 50mM RD RK MEHQ pHPA 2pHP-IA pHPE or 100mM MEHQ and pHPA individually andfor measuring the survival rates the medium was replacedwithDMEMcontaining 0165mgmLneutral red After beingcultured for 2 h the cells were washed with phosphatebuffered saline (PBS) and neutral red incorporated in thecells was extracted with NR extraction solution which wasa mixture of methanol acetic acid and water at a ratioof 50 1 49 The absorbance of neutral red in a well was







2O2wasmeasured





2O2(30mM)


2PO4at a




3 Results



0

20

40

60

80

100

120

RD RK

MEH

Q

pHPA

2pH

P-IA

pHPE

MEH

Q

pHPA

Surv

ival

rate

()

HaCaTB16F10

50 mM 100mM



lowastlowast

lowastlowast



















4 Discussion










Con

trol B

16F1

0B1

6F1

0+10

mM

RK


00

10

20

30


Relat

ive i

ntra

cellu

lar R

OS

leve

l

HaCaTB16F10

lowastlowast

lowastlowast

lowastlowast

01mM






00

05

10

15

20

25

30

35

40 60 80 100

Relat

ive i

ntra

cellu

lar R

OS

leve

l

Survival rate ()

y = minus00351x + 43578

r = minus0888

P = 0018

(a)

00

05

10

15

20

60 70 80 90 100

Relat

ive i

ntra

cellu

lar R

OS

leve

l

Survival rate ()

y = minus00064x + 05358

r = 0377

P = 0461

(b)


00

10

20

30

40

50

60

70

0 500 750 1000

Relat

ive i

ntra

cellu

lar R

OS

leve

l


0mM RK10mM RK

lowastlowast

lowastlowast






0mM RK10mM RK


00

10

20

30

40

50

0 025 050 100

Relat

ive i

ntra

cellu

lar R

OS

leve

l

EDTA (mM)


00

05

10

15

20

Ctl RD RK

Rela

tive i

ntra

cellu

lar H

2O2

leve

l

lowast lowast

50mM








(a) H2O2








2O2







2O2


2O2[38]













00

20

40

60

80

100

Relat

ive E

SR p

eak

inte

nsity

val

ue

RD

200 500 1000


UVB (Jm2)


lowastlowast




58

0

50

100

150

200

Peak

inte

nsity

(mAU

)


20

(a) 0min

58

55 64

0

50

100

Peak

inte

nsity

(mAU

)

4750


20

(b) 15min

55

40 60

50

100

Peak

inte

nsity

(mAU

)


0

20

(c) 30min



HO HO O

OHO HO

HO

O

OH OH

OH

OH

OH

Tyrosinase

(a) (b)

(c)

(d)

Tyrosinase+

H2O2

O2

Tyrosinase+O2

∙

∙

O

(c998400)OH∙



5 Conclusions



2O2








Acknowledgments


References

















































Volume 2014

ToxinsJournal of

VaccinesJournal of















AddictionJournal of






Autoimmune Diseases




Journal of


Pharmaceutics



of







2O2wasmeasured





2O2(30mM)


2PO4at a




3 Results



0

20

40

60

80

100

120

RD RK

MEH

Q

pHPA

2pH

P-IA

pHPE

MEH

Q

pHPA

Surv

ival

rate

()

HaCaTB16F10

50 mM 100mM



lowastlowast

lowastlowast



















4 Discussion










Con

trol B

16F1

0B1

6F1

0+10

mM

RK


00

10

20

30


Relat

ive i

ntra

cellu

lar R

OS

leve

l

HaCaTB16F10

lowastlowast

lowastlowast

lowastlowast

01mM






00

05

10

15

20

25

30

35

40 60 80 100

Relat

ive i

ntra

cellu

lar R

OS

leve

l

Survival rate ()

y = minus00351x + 43578

r = minus0888

P = 0018

(a)

00

05

10

15

20

60 70 80 90 100

Relat

ive i

ntra

cellu

lar R

OS

leve

l

Survival rate ()

y = minus00064x + 05358

r = 0377

P = 0461

(b)


00

10

20

30

40

50

60

70

0 500 750 1000

Relat

ive i

ntra

cellu

lar R

OS

leve

l


0mM RK10mM RK

lowastlowast

lowastlowast






0mM RK10mM RK


00

10

20

30

40

50

0 025 050 100

Relat

ive i

ntra

cellu

lar R

OS

leve

l

EDTA (mM)


00

05

10

15

20

Ctl RD RK

Rela

tive i

ntra

cellu

lar H

2O2

leve

l

lowast lowast

50mM








(a) H2O2








2O2







2O2


2O2[38]













00

20

40

60

80

100

Relat

ive E

SR p

eak

inte

nsity

val

ue

RD

200 500 1000


UVB (Jm2)


lowastlowast




58

0

50

100

150

200

Peak

inte

nsity

(mAU

)


20

(a) 0min

58

55 64

0

50

100

Peak

inte

nsity

(mAU

)

4750


20

(b) 15min

55

40 60

50

100

Peak

inte

nsity

(mAU

)


0

20

(c) 30min



HO HO O

OHO HO

HO

O

OH OH

OH

OH

OH

Tyrosinase

(a) (b)

(c)

(d)

Tyrosinase+

H2O2

O2

Tyrosinase+O2

∙

∙

O

(c998400)OH∙



5 Conclusions



2O2








Acknowledgments


References

















































Volume 2014

ToxinsJournal of

VaccinesJournal of















AddictionJournal of






Autoimmune Diseases




Journal of


Pharmaceutics



of


0

20

40

60

80

100

120

RD RK

MEH

Q

pHPA

2pH

P-IA

pHPE

MEH

Q

pHPA

Surv

ival

rate

()

HaCaTB16F10

50 mM 100mM



lowastlowast

lowastlowast



















4 Discussion










Con

trol B

16F1

0B1

6F1

0+10

mM

RK


00

10

20

30


Relat

ive i

ntra

cellu

lar R

OS

leve

l

HaCaTB16F10

lowastlowast

lowastlowast

lowastlowast

01mM






00

05

10

15

20

25

30

35

40 60 80 100

Relat

ive i

ntra

cellu

lar R

OS

leve

l

Survival rate ()

y = minus00351x + 43578

r = minus0888

P = 0018

(a)

00

05

10

15

20

60 70 80 90 100

Relat

ive i

ntra

cellu

lar R

OS

leve

l

Survival rate ()

y = minus00064x + 05358

r = 0377

P = 0461

(b)


00

10

20

30

40

50

60

70

0 500 750 1000

Relat

ive i

ntra

cellu

lar R

OS

leve

l


0mM RK10mM RK

lowastlowast

lowastlowast






0mM RK10mM RK


00

10

20

30

40

50

0 025 050 100

Relat

ive i

ntra

cellu

lar R

OS

leve

l

EDTA (mM)


00

05

10

15

20

Ctl RD RK

Rela

tive i

ntra

cellu

lar H

2O2

leve

l

lowast lowast

50mM








(a) H2O2








2O2







2O2


2O2[38]













00

20

40

60

80

100

Relat

ive E

SR p

eak

inte

nsity

val

ue

RD

200 500 1000


UVB (Jm2)


lowastlowast




58

0

50

100

150

200

Peak

inte

nsity

(mAU

)


20

(a) 0min

58

55 64

0

50

100

Peak

inte

nsity

(mAU

)

4750


20

(b) 15min

55

40 60

50

100

Peak

inte

nsity

(mAU

)


0

20

(c) 30min



HO HO O

OHO HO

HO

O

OH OH

OH

OH

OH

Tyrosinase

(a) (b)

(c)

(d)

Tyrosinase+

H2O2

O2

Tyrosinase+O2

∙

∙

O

(c998400)OH∙



5 Conclusions



2O2








Acknowledgments


References

















































Volume 2014

ToxinsJournal of

VaccinesJournal of















AddictionJournal of






Autoimmune Diseases




Journal of


Pharmaceutics



of





4 Discussion










Con

trol B

16F1

0B1

6F1

0+10

mM

RK


00

10

20

30


Relat

ive i

ntra

cellu

lar R

OS

leve

l

HaCaTB16F10

lowastlowast

lowastlowast

lowastlowast

01mM






00

05

10

15

20

25

30

35

40 60 80 100

Relat

ive i

ntra

cellu

lar R

OS

leve

l

Survival rate ()

y = minus00351x + 43578

r = minus0888

P = 0018

(a)

00

05

10

15

20

60 70 80 90 100

Relat

ive i

ntra

cellu

lar R

OS

leve

l

Survival rate ()

y = minus00064x + 05358

r = 0377

P = 0461

(b)


00

10

20

30

40

50

60

70

0 500 750 1000

Relat

ive i

ntra

cellu

lar R

OS

leve

l


0mM RK10mM RK

lowastlowast

lowastlowast






0mM RK10mM RK


00

10

20

30

40

50

0 025 050 100

Relat

ive i

ntra

cellu

lar R

OS

leve

l

EDTA (mM)


00

05

10

15

20

Ctl RD RK

Rela

tive i

ntra

cellu

lar H

2O2

leve

l

lowast lowast

50mM








(a) H2O2








2O2







2O2


2O2[38]













00

20

40

60

80

100

Relat

ive E

SR p

eak

inte

nsity

val

ue

RD

200 500 1000


UVB (Jm2)


lowastlowast




58

0

50

100

150

200

Peak

inte

nsity

(mAU

)


20

(a) 0min

58

55 64

0

50

100

Peak

inte

nsity

(mAU

)

4750


20

(b) 15min

55

40 60

50

100

Peak

inte

nsity

(mAU

)


0

20

(c) 30min



HO HO O

OHO HO

HO

O

OH OH

OH

OH

OH

Tyrosinase

(a) (b)

(c)

(d)

Tyrosinase+

H2O2

O2

Tyrosinase+O2

∙

∙

O

(c998400)OH∙



5 Conclusions



2O2








Acknowledgments


References

















































Volume 2014

ToxinsJournal of

VaccinesJournal of















AddictionJournal of






Autoimmune Diseases




Journal of


Pharmaceutics



of



Con

trol B

16F1

0B1

6F1

0+10

mM

RK


00

10

20

30


Relat

ive i

ntra

cellu

lar R

OS

leve

l

HaCaTB16F10

lowastlowast

lowastlowast

lowastlowast

01mM






00

05

10

15

20

25

30

35

40 60 80 100

Relat

ive i

ntra

cellu

lar R

OS

leve

l

Survival rate ()

y = minus00351x + 43578

r = minus0888

P = 0018

(a)

00

05

10

15

20

60 70 80 90 100

Relat

ive i

ntra

cellu

lar R

OS

leve

l

Survival rate ()

y = minus00064x + 05358

r = 0377

P = 0461

(b)


00

10

20

30

40

50

60

70

0 500 750 1000

Relat

ive i

ntra

cellu

lar R

OS

leve

l


0mM RK10mM RK

lowastlowast

lowastlowast






0mM RK10mM RK


00

10

20

30

40

50

0 025 050 100

Relat

ive i

ntra

cellu

lar R

OS

leve

l

EDTA (mM)


00

05

10

15

20

Ctl RD RK

Rela

tive i

ntra

cellu

lar H

2O2

leve

l

lowast lowast

50mM








(a) H2O2








2O2







2O2


2O2[38]













00

20

40

60

80

100

Relat

ive E

SR p

eak

inte

nsity

val

ue

RD

200 500 1000


UVB (Jm2)


lowastlowast




58

0

50

100

150

200

Peak

inte

nsity

(mAU

)


20

(a) 0min

58

55 64

0

50

100

Peak

inte

nsity

(mAU

)

4750


20

(b) 15min

55

40 60

50

100

Peak

inte

nsity

(mAU

)


0

20

(c) 30min



HO HO O

OHO HO

HO

O

OH OH

OH

OH

OH

Tyrosinase

(a) (b)

(c)

(d)

Tyrosinase+

H2O2

O2

Tyrosinase+O2

∙

∙

O

(c998400)OH∙



5 Conclusions



2O2








Acknowledgments


References

















































Volume 2014

ToxinsJournal of

VaccinesJournal of















AddictionJournal of






Autoimmune Diseases




Journal of


Pharmaceutics



of


00

05

10

15

20

25

30

35

40 60 80 100

Relat

ive i

ntra

cellu

lar R

OS

leve

l

Survival rate ()

y = minus00351x + 43578

r = minus0888

P = 0018

(a)

00

05

10

15

20

60 70 80 90 100

Relat

ive i

ntra

cellu

lar R

OS

leve

l

Survival rate ()

y = minus00064x + 05358

r = 0377

P = 0461

(b)


00

10

20

30

40

50

60

70

0 500 750 1000

Relat

ive i

ntra

cellu

lar R

OS

leve

l


0mM RK10mM RK

lowastlowast

lowastlowast






0mM RK10mM RK


00

10

20

30

40

50

0 025 050 100

Relat

ive i

ntra

cellu

lar R

OS

leve

l

EDTA (mM)


00

05

10

15

20

Ctl RD RK

Rela

tive i

ntra

cellu

lar H

2O2

leve

l

lowast lowast

50mM








(a) H2O2








2O2







2O2


2O2[38]













00

20

40

60

80

100

Relat

ive E

SR p

eak

inte

nsity

val

ue

RD

200 500 1000


UVB (Jm2)


lowastlowast




58

0

50

100

150

200

Peak

inte

nsity

(mAU

)


20

(a) 0min

58

55 64

0

50

100

Peak

inte

nsity

(mAU

)

4750


20

(b) 15min

55

40 60

50

100

Peak

inte

nsity

(mAU

)


0

20

(c) 30min



HO HO O

OHO HO

HO

O

OH OH

OH

OH

OH

Tyrosinase

(a) (b)

(c)

(d)

Tyrosinase+

H2O2

O2

Tyrosinase+O2

∙

∙

O

(c998400)OH∙



5 Conclusions



2O2








Acknowledgments


References

















































Volume 2014

ToxinsJournal of

VaccinesJournal of















AddictionJournal of






Autoimmune Diseases




Journal of


Pharmaceutics



of


0mM RK10mM RK


00

10

20

30

40

50

0 025 050 100

Relat

ive i

ntra

cellu

lar R

OS

leve

l

EDTA (mM)


00

05

10

15

20

Ctl RD RK

Rela

tive i

ntra

cellu

lar H

2O2

leve

l

lowast lowast

50mM








(a) H2O2








2O2







2O2


2O2[38]













00

20

40

60

80

100

Relat

ive E

SR p

eak

inte

nsity

val

ue

RD

200 500 1000


UVB (Jm2)


lowastlowast




58

0

50

100

150

200

Peak

inte

nsity

(mAU

)


20

(a) 0min

58

55 64

0

50

100

Peak

inte

nsity

(mAU

)

4750


20

(b) 15min

55

40 60

50

100

Peak

inte

nsity

(mAU

)


0

20

(c) 30min



HO HO O

OHO HO

HO

O

OH OH

OH

OH

OH

Tyrosinase

(a) (b)

(c)

(d)

Tyrosinase+

H2O2

O2

Tyrosinase+O2

∙

∙

O

(c998400)OH∙



5 Conclusions



2O2








Acknowledgments


References

















































Volume 2014

ToxinsJournal of

VaccinesJournal of















AddictionJournal of






Autoimmune Diseases




Journal of


Pharmaceutics



of


(a) H2O2








2O2







2O2


2O2[38]













00

20

40

60

80

100

Relat

ive E

SR p

eak

inte

nsity

val

ue

RD

200 500 1000


UVB (Jm2)


lowastlowast




58

0

50

100

150

200

Peak

inte

nsity

(mAU

)


20

(a) 0min

58

55 64

0

50

100

Peak

inte

nsity

(mAU

)

4750


20

(b) 15min

55

40 60

50

100

Peak

inte

nsity

(mAU

)


0

20

(c) 30min



HO HO O

OHO HO

HO

O

OH OH

OH

OH

OH

Tyrosinase

(a) (b)

(c)

(d)

Tyrosinase+

H2O2

O2

Tyrosinase+O2

∙

∙

O

(c998400)OH∙



5 Conclusions



2O2








Acknowledgments


References

















































Volume 2014

ToxinsJournal of

VaccinesJournal of















AddictionJournal of






Autoimmune Diseases




Journal of


Pharmaceutics



of


00

20

40

60

80

100

Relat

ive E

SR p

eak

inte

nsity

val

ue

RD

200 500 1000


UVB (Jm2)


lowastlowast




58

0

50

100

150

200

Peak

inte

nsity

(mAU

)


20

(a) 0min

58

55 64

0

50

100

Peak

inte

nsity

(mAU

)

4750


20

(b) 15min

55

40 60

50

100

Peak

inte

nsity

(mAU

)


0

20

(c) 30min



HO HO O

OHO HO

HO

O

OH OH

OH

OH

OH

Tyrosinase

(a) (b)

(c)

(d)

Tyrosinase+

H2O2

O2

Tyrosinase+O2

∙

∙

O

(c998400)OH∙



5 Conclusions



2O2








Acknowledgments


References

















































Volume 2014

ToxinsJournal of

VaccinesJournal of















AddictionJournal of






Autoimmune Diseases




Journal of


Pharmaceutics



of


HO HO O

OHO HO

HO

O

OH OH

OH

OH

OH

Tyrosinase

(a) (b)

(c)

(d)

Tyrosinase+

H2O2

O2

Tyrosinase+O2

∙

∙

O

(c998400)OH∙



5 Conclusions



2O2








Acknowledgments


References

















































Volume 2014

ToxinsJournal of

VaccinesJournal of















AddictionJournal of






Autoimmune Diseases




Journal of


Pharmaceutics



of





































Volume 2014

ToxinsJournal of

VaccinesJournal of















AddictionJournal of






Autoimmune Diseases




Journal of


Pharmaceutics



of

research article the mechanism of melanocytes-specific...

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