Original Article GenerationJCBNJournal0912-00091880-5086theKyoto,jcbn16-3810.3164/jcbn.16-38Original Society Japanof Article Clinical for Free Biochemistry Radical Research of and Nutrition hydroxylJapan radicals and singlet oxygen during oxidation of rhododendrol and rhododendrolcatechol Akimitsu Miyaji,1 Yu Gabe,2 Masahiro Kohno3 and Toshihide Baba1,* 1Department of Environmental Chemistry and Engineering, Tokyo Institute of Technology, 4259G114, Nagatsutacho, Midoriku, Yokohama 2268502, Japan 2Biological Science Laboratories, Kao Corporation, 2606 Akabane, Ichikaimachi, Hagagun, Tochigi 3213497, Japan 3Department of Bioengineering, Tokyo Institute of Technology, 4259G125, Nagatsutacho, Midoriku, Yokohama 2268502, Japan (Received3 31 March, 2016; Accepted 10 July, 2016; Published online 5 October, 2016) CreativestrictedvidedTheCopyright2017This generation the isuse, originalanCommons opendistribution,© of2017 workhydroxylaccess JCBNAttribution is article andproperly radicals reproduction distributed License, cited.and singlet underwhichin anyoxygen the permitsmedium, terms during ofunre- pro- the rhododendrol seems to be due to competition between rhododendrol oxidation of 4(4hydroxyphenyl)2butanol (rhododendrol) and oxidation and L-tyrosine oxidation at tyrosinase active sites. 4(3,4dihydroxyphenyl)2butanol (rhododendrolcatechol) with 4-(3-hydroxybutyl)-1,2-benzoquinone (rhododendrol quinone), mushroom tyrosinase in a phosphate buffer (pH 7.4) was examined 4-(3,4-dihydroxyphenyl)-2-butanol (rhododendrol-catechol), 6,7- as the model for the reactive oxygen species generation via the two dihydroxy-2-methylchromane (Cyclic rhododendrol-catechol), and rhododendrol compounds in melanocytes. The reaction was per 5-(3-hydroxybutyl)-benzene-1,2,4-triol (hydroxyl rhododendrol- formed in the presence of 5,5dimethyl1pyrrolineNoxide (DMPO) catechol) have been identified as reaction products.(9) Rhododendrol- spin trap reagents for hydroxyl radical or 2,2,6,6tetramethyl4 quionone is the product of tyrosinase-catalyzed rhododendrol piperidone (4oxoTEMP), an acceptor of singlet oxygen, and their oxidation as shown in Fig. 1A. Cyclic rhododendrol-catechol is electron spin resonances were measured. An increase in the produced by the cyclization of rhododendrol-quinone as shown in electron spin resonances signal attributable to the adduct of Fig. 1B, which is similar to cyclization of dopaquionone to DMPO reacting with the hydroxyl radical and that of 4oxoTEMP leukodopachrome in the synthesis of melanin from L-tyrosine.(11) reacting with singlet oxygen was observed during the tyrosinase Hydroxyl rhododendrol-catechol is probably produced by hydroxy- catalyzed oxidation of rhododendrol and rhododendrolcatechol, lation of rhododendrol-quinone as shown in Fig. 1C. Rhododendrol- indicating the generation of hydroxyl radical and singlet oxygen. catechol may be produced by the reaction of rhododendrol- Moreover, hydroxyl radical generation was also observed in the quinone with other catechol-derivatives as shown in Fig. 1D and autoxidation of rhododendrolcatechol. We show that generation E. This latter reaction is predicted based on L-dopa generation in of intermediates during tyrosinasecatalyzed oxidation of rhodo tyrosinase-catalyzed L-tyrosine oxidation in melanogenesis.(11) dendrol enhances oxidative stress in melanocytes. Rhododendrol-catechol is also oxidized by tyrosinase as shown in Fig. 1F. Rhododenrol-catechol shows higher toxicity in cells Key Words: autoxidation, hydroxyl radical, rhododendrol, than rhododendrol.(7,8) singlet oxygen, tyrosinase One of the reasons for leukoderma caused by rhododendrol is thought to be oxidative stress induced by tyrosinase-catalyzed IntroductionThe generation of reactive oxygen species (ROS) during oxidation.(12) In fact, rhododendrol increased the level of intra- T tyrosinase-catalyzed oxidation reactions has been reported cellular ROS and hydrogen peroxide in melanoma cells. The previously.(1) The generation of hydrogen peroxide and superoxide generation of hydroxyl radicals was observed in a mixture of anions during the oxidation of L-tyrosine in the presence of rhododendrol, tyrosinase, and hydrogen peroxide, and the genera- tyrosinase was observed using a chemiluminescence probe.(2) The tion was enhanced by irradiation with 1,000 J/m2 UV-B (ultraviolet generation of hydroxyl radicals during the oxidation of L-tyrosine light with 280–315 nm-wavelength).(13) Further, ROS generation and L-dopa in the presence of tyrosinase was confirmed using was detected during rhododendrol oxidation by tyrosinase.(14) an electron spin resonance (ESR) spin trapping reagent, 5,5- Hence, in addition to hydrogen peroxide, hydroxyl radicals and (3) 1 dimethyl-1-pyrroline-N-oxide (DMPO). These results suggest singlet oxygen ( O2) are strong candidates for the onset of that ROS generation occurs in melanocytes, the cells synthesizing leukoderma caused by rhododendrol. melanin in which tyrosinase operates. The aim of this study is to study the generation of hydroxyl 1 Once the balance of ROS generation and the antioxidant radicals and O2 during the oxidation of rhododendrol and capacity in melanocytes is disturbed, excess ROS in melanocytes rhododendrol-catechol. We measured the amount of ROS present causes a decrease in the antioxidant capacity due to oxidative during the oxidation that occurred at a pH of ~7 so that the oxida- stress.(4) ROS damage DNA, proteins, and lipids in cells and, tive stress caused by the imbalance of ROS generation and anti- thus, are responsible for the pathogenesis of numerous diseases. oxidant capacity in biological systems could be investigated. We Oxidative stress is thought to be involved in the development of applied the electron spin resonance (ESR) spin-trapping method (5) 1 skin diseases such as melanoma. to measure the generation of hydroxyl radicals and O2 during Recently, those who used lightening and whitening cosmetics rhododendrol oxidation. A hydrophilic spin-trapping probe working containing 4-(4-hydroxyphenyl)-2-butanol (rhododendrol) suffered at a pH of ~7 was used under conditions needed for carrying from leukoderma,(6) a cutaneous condition with localized loss of out enzymatic reactions. Thus, 5,5-dimethyl-1-pyrroline-N-oxide pigmentation. Rhododendrol can cause tyrosinase-related toxicity (7,8) in cells. Rhododendrol is converted to catechol- and o-quinone *To whom correspondence should be addressed. (9,10) derivatives. Reduction of melanin synthesis in melanocytes by Email: [email protected] doi: 10.3164/jcbn.1638 J. Clin. Biochem. Nutr. | March 2017 | vol. 60 | no. 2 | 86–92 ©2017 JCBN Fig. 1. Rhododendrol oxidation. (DMPO) was used as the probe for hydroxyl radicals,(15) and filtered reaction mixture was diluted 5 times with a 30-mM 2,2,6,6-tetramethyl-4-piperidone hydrochloride (4-oxo-TEMP) phosphate buffer (pH 7.4). The diluted reaction mixture was then 1 (16) was used for O2 detection. These probes have been applied transferred to a quartz cell (light path length of 1 cm). Its absorp- previously in biological systems with successful detection of tion spectrum for wavelengths of 200–800 nm was measured ROS.(3,17) using a Multispec-1500 spectral measurement system (Shimadzu, Kyoto, Japan). 1 Materials and Methods Detection of hydroxyl radicals and O2 using ESR. The reaction mixture was transferred to a quartz flat cell (150 μl) Oxidation of rhododendrol and rhododendrolcatechol in for ESR measurements. The optical path length was 0.25 mm. the presence of a spintrap reagent for hydroxyl radical or an The ESR spectra of the various samples were recorded at room acceptor of singlet oxygen. Rhododendrol and rhododendrol- temperature using an X-band spectrometer (JES-FA-100, JEOL, catechol were kindly provided from Kao Corporation (Tokyo, Tokyo, Japan), which was operated at 9.43 GHz. The magnetic Japan). The oxidation of rhododendrol and rhododendrol-catechol field was modulated at 100 kHz. The conditions for measurement in the presence of tyrosinase (from mushrooms; Sigma-Aldrich, of ESR spectra were as follows: microwave power of 4 mW; Tokyo, Japan) was performed in the presence of a spin-trap magnetic field of 335.5 ± 5.0 mT; field modulation width of reagent in a water bath at 37°C. A spin-trap reagent, DMPO 0.1 mT; sweep time of 2 min; time constant of 0.1 s. (Labotech, Tokyo, Japan), was used to detect hydroxyl radicals The signal intensities were normalized with respect to a MnO and superoxide anions, while 4-oxo-TEMP (Sigma-Aldrich) was marker, and the concentrations of the stable radical products were 1 used for detecting O2. The appropriate reagent for detecting determined on the basis of the signal height using an external 1 hydroxyl radical or O2 and rhododendrol or rhododendrol- standard, 2,2,6,6-teteramethyl piperidinol (TEMPOL; Sigma- catechol were dissolved in a 30 mM phosphate buffer (pH 7.4). Aldrich). Tyrosinase was added to this solution in an amount of 100 units Estimation of bond dissociation energies of the oxygen (U)/ml to initiate the enzymatic oxidation reaction. The value of hydrogen bond in catechol derivatives. Catechol- and phenol- one enzyme unit of commercial tyrosinase was provided by the derivative models were constructed and optimized using density supplier, Sigma-Aldrich. Autoxidation of rhododendrol-catechol functional theory (DFT). The long-range and dispersion corrected was carried out under the same reaction conditions described ωB97X-D functional with 6-311G(d,p) basis sets were selected.(18–20) above except for the