eumelanin is enhanced by ultraviolet A radiation - Wiley Online Library

DR. SHOSUKE ITO (Orcid ID : 0000-0001-9182-5144)

Accepted Article

PROF. KAZUMASA WAKAMATSU (Orcid ID : 0000-0003-1748-9001) Article type

: Short Communication

Manuscript Category: Melanin Chemistry & Pigmentation (MCP)

The potent pro-oxidant activity of rhododendrol-eumelanin is enhanced by ultraviolet A radiation

Shosuke Ito, Misa Agata, Kotono Okochi and Kazumasa Wakamatsu Department of Chemistry, Fujita Health University School of Health Sciences, Toyoake, Aichi, Japan

*Address correspondence to: Dr. Shosuke Ito Department of Chemistry Fujita Health University School of Health Sciences Toyoake, Aichi 470-1192, Japan TEL: +81 562 93 2595 FAX: +81 562 93 4595

This article has been accepted for publication and undergone full peer review but has not been through the copyediting, typesetting, pagination and proofreading process, which may lead to differences between this version and the Version of Record. Please cite this article as doi: 10.1111/pcmr.12696 This article is protected by copyright. All rights reserved.

Accepted Article

Email: [email protected]

Summary RS-4-(4-hydroxyphenyl)-2-butanol (rhododendrol, RD), a skin-whitening agent, is known to induce leukoderma in some consumers. To explore the mechanism underlying this effect, we previously showed that the oxidation of RD with mushroom or human tyrosinase produces cytotoxic quinone oxidation products and RD-eumelanin exerts a potent pro-oxidant activity. Cellular antioxidants were oxidized by RD-eumelanin with a concomitant production of H2O2. In this study, we examined whether this pro-oxidant activity of RD-eumelanin is enhanced by ultraviolet A (UVA) radiation because most RD-induced leukoderma lesions are found in sun-exposed areas. Exposure to a physiological level of UVA (3.5 mW/cm2) induced a two to four-fold increase in the rates of oxidation of GSH, cysteine, ascorbic acid and NADH. This oxidation was oxygen-dependent and was accompanied by the production of H2O2. These results suggest that RD-eumelanin is cytotoxic to melanocytes through its potent pro-oxidant activity that is enhanced by UVA radiation.

Significance Topical application with rhododendrol (RD) induces leukoderma in the skin of some consumers. Quinone oxidation products from RD rapidly polymerize to form RD-eumelanin. This oligomeric pigment is redox-active, oxidizing cellular antioxidants to deplete them and reducing molecular oxygen to generate reactive oxygen species. This pro-oxidant activity of RD-eumelanin is enhanced by UVA radiation, thus raising the possibility that RD-induced leukoderma is exacerbated by UVA radiation. This could possibly be related to the fact that the incidence of this RD-induced leukoderma occurs more frequently in the summer season.

Keywords: antioxidants, hydrogen peroxide, pro-oxidant, rhododendrol, ultraviolet A

This article is protected by copyright. All rights reserved.

RS-4-(4-hydroxyphenyl)-2-butanol (rhododendrol, also called rhododenol [RD], 1 in Figure 1) had

Accepted Article

been added to cosmetics as a skin-whitening ingredient by a cosmetic company in Japan. In July 2013, cosmetics containing RD were recalled by that company because a considerable number of consumers developed leukoderma on their face, neck and hands. Because skin biopsy samples taken from depigmented lesions of affected patients showed few or no detectable melanocytes, it was suggested that RD was cytotoxic to melanocytes under certain conditions. In fact, RD was shown to exert its specific toxicity to melanocytes via a tyrosinase-dependent mechanism (Kasamatsu et al., 2014; Sasaki et al., 2014). We also found that the oxidation of racemic RD (1) by mushroom tyrosinase rapidly produces RD-quinone (2), which is quickly converted to RD-hydroxy-p-quinone (3) through addition of water molecule (Ito et al., 2014a). We then confirmed that human tyrosinase is able to oxidize both enantiomers of RD (Ito et al., 2014b). We also showed that RD-catechol was much more toxic to melanocytes than RD (Okura et al., 2015). Subsequently, RD-pheomelanin and its precursor cysteinyl-RD-catechol were detected in

murine B16F1 melanoma cells exposed to RD (Ito et al., 2015). In a more recent study (Ito et al., 2017), we compared the pro-oxidant activities of synthetic RD-eumelanin (RD-EM), RD-pheomelanin, Dopa-eumelanin and Dopa-pheomelanin. In this regard, it is known that pheomelanin (Dopa-pheomelanin) exerts a strong pro-oxidant activity in yellow mice that can lead to melanomagenesis (Mitra et al., 2012; Morgan et al., 2013). That in vivo observation was supported by subsequent biochemical studies showing the facile oxidation (depletion) of GSH and NADH by red hair pheomelanin (Napolitano et al., 2014; Panzella et al., 2014). A study by Ito et al. (2017) showed that synthetic RD-eumelanin (RD-EM) is a pro-oxidant as potent as synthetic Dopa-pheomelanin (Dopa-PM) in oxidizing GSH, cysteine (CySH), ascorbic acid (AA) and NADH with a concomitant production of reactive oxygen species (ROS). RD-EM is thus redox-active, possessing both oxidizing and reducing activities. This redox property has been known for many years for melanins (Korytowski, et al., 1986; 1987), and a recent study by Kim et al. (2015a) demonstrated that both pheomelanin and eumelanin are redox-active, that they can rapidly and repeatedly redox-cycle between oxidized and reduced states, and that pheomelanin possesses a more oxidative redox potential than eumelanin. In this study, we examined whether this pro-oxidant activity of RD-EM is enhanced by

ultraviolet A (UVA) radiation because most RD-induced leukoderma lesions were found in sun-exposed areas (Aoyama et al., 2014). That study also showed that RD-induced leukoderma


occurred more frequently in the summer months compared to the spring and autumn months (Aoyama

Accepted Article

et al., 2014). We found that exposure to a physiological level of UVA (3.5 mW/cm2; Haywood et al., 2011) induces two to four-fold increases in the rates of oxidation of GSH, CySH, AA and NADH. This oxidation is oxygen-dependent and is accompanied by the production of H2O2. These results suggest that RD-eumelanin induces cytotoxicity through its potent pro-oxidant activity that is enhanced by UVA radiation. RD-EM was prepared by oxidizing 100 M RD with mushroom tyrosinase at pH 7.4 for 120

min. This 120 min period was found to be sufficient to oxidize and polymerize RD to an oligomeric oxidation product RD-EM (Ito et al., 2014a; Ito et al., 2017). This RD-EM was mixed with 10 molar eq. of antioxidants, GSH, CySH, AA and NADH (final concentrations: antioxidant 910 M and RD-EM 91 M). The use of a 10 molar excess of antioxidant was to confirm that the oxidation of antioxidants (and the reduction of molecular oxygen) proceeds through the catalytic action of RD-EM. Two cuvettes containing a solution of the mixture of RD-EM and an antioxidant were irradiated with UVA at room temperature (27-28˚C) while two other cuvettes containing the same solution were protected from UVA exposure with aluminum foil (no UVA control). The oxidation was followed every 30 min up to 120 min by measuring the remaining antioxidant using HPLC (Imai et al., 1987; Ito et al., 2017). As shown in Figure 2A, GSH levels decreased gradually and continuously during 120 min, a

rate that was much faster with UVA irradiation. To determine the degree of enhancement by UVA, we analyzed the rate of oxidation by zero-order and first-order kinetics. The GSH oxidation was enhanced 4.2-fold by UVA radiation (Figure S1A, B; Table 1). The oxidation of GSH to oxidized glutathione (GSSG) was not examined in the present study. However, in a previous study (Ito et al., 2017), the decrease of GSH during autoxidation was found to be mostly due to the oxidation to GSSG. CySH levels decreased 3 to 4-fold faster than GSH levels (Figure 2B). The CySH oxidation was enhanced 2.0-fold by UVA radiation (Figure S1C, D, Table 1). The control experiments showed that in the absence of added RD-EM, the oxidation of CySH proceeded much slower (about one-fifth) than that in its presence (Figure S2A, B and Table 1). We then examined cellular antioxidants other than GSH and CySH for oxidation by RD-EM. As shown in Figure 2C, AA was rapidly oxidized by RD-EM and the oxidation was enhanced by UVA irradiation. The AA oxidation was enhanced


2.4-fold by UVA radiation (Figure S1E, F; Table 1). The control experiments showed that the

Accepted Article

oxidation of AA in the absence of RD-EM proceeded much slower (less than one-tenth; Table 1). NADH was also oxidized at rates comparable to GSH (Figure 2D). The NADH oxidation was enhanced 3.9-fold by UVA radiation (Figure S1G, H; Table 1). NADH absorbs UVA strongly with a maximum at 340 nm. Therefore, the direct photo-oxidation of NADH in the absence of RD-EM was also examined. The results indicate that NADH was oxidized more slowly with a rate ca. half that in the presence of RD-EM (Figure S2C, D; Table 1). Thus, an additional effect of RD-EM in the NADH oxidation was confirmed. Next, we examined the roles of oxygen in the potent pro-oxidant activity of RD-EM. The

involvement of molecular oxygen was examined first. The oxidation of CySH (10 molar eq.) by RD-EM was compared in the presence or absence of air with/without UVA radiation for 120 min. We purged the air using an argon stream. As shown in Figure 3A, the CySH oxidation was significantly suppressed in the absence of oxygen to 46% (from 91% to 42%, with UVA radiation) and 36% (from 51% to 18%, with no radiation) as compared to the presence of air (oxygen). We then examined whether H2O2 is produced during the oxidation of antioxidants by RD-EM. H2O2 was analyzed by the method of Zhou et al. (1997) using the chromogen Ampliflu Red reagent (1-acetyl-3,7-dihydroxyphenoxazine). After the oxidation of GSH, CySH and AA (910 M) by RD-EM (91 M) for 120 min, 10 to 40 M H2O2 was detected (Figure 3B). The production of H2O2 was slightly greater with UVA radiation, although the difference was not significant. Moreover, the level of H2O2 was much lower than expected from the oxidation of antioxidants. However, the NADH oxidation produced a 3.6-fold greater level of H2O2 (129 M) compared to the non-irradiated control (36 M) and the other antioxidants. We next examined whether H2O2 can be reduced by the antioxidants used in this study to confirm that the H2O2 produced during the oxidation of antioxidants is decomposed by the antioxidants themselves. As shown in Figure 3C, H2O2 (910 M) was reduced by different antioxidants (455 M) at various rates. AA was most efficient in reducing 89% of H2O2, which was followed by CySH (80%), GSH (60%) and NADH (29%). These results indicate that H2O2 should have been produced at levels much greater than those shown in Figure 3B. In this regard, it is known that ROS generation is significantly increased in B16 melanoma cells after exposure to RD (Kim et al., 2016; Nagata et al., 2015; Okura et al., 2015).


In this study, we examined whether the pro-oxidant activity of RD-EM is enhanced by

Accepted Article

irradiation with UVA light. We found that exposure to a physiological level of UVA (3.5 mW/cm2) induces a two to four-fold increase in the rates of oxidation of GSH, CySH, AA and NADH. This oxidation is oxygen-dependent and is accompanied by the production of H2O2. In our previous study (Ito et. al., 2017), the tyrosinase-catalyzed oxidation of RD was found to produce a dimeric form of quinoid oxidation products (Figure 1) as early as 10 min, which peaked at 120 min with a gradual shift to possibly a tetrameric form. We designated this mixture of quinoid oxidation products as RD-EM. The oxidized form of RD-EM (Figure 1) is able to oxidize a variety of antioxidants (Ito et al., 2017). This reaction mimics the oxidation of thiols by the benzothiazine moiety of pheomelanin yielding disulfides accompanied by the production of the dihydrobenzothiazine moiety (Napolitano et al., 2014; Panzella et al., 2014). Napolitano et al. (2014) proposed that this reaction proceeds with a one electron transfer from the thiol to the benzothiazine moiety. A similar mechanism may operate in the oxidized form of RD-EM. The present study shows that the oxidation of antioxidants by the oxidized RD-EM is accelerated by UVA radiation (Figure 1). The oxidation of antioxidants accompanies the production of the reduced form of RD-EM

(Figure 1). This form of RD-EM is able to reduce molecular oxygen to form superoxide radicals and this process is accelerated by UVA radiation. It has been known for some years that superoxide radicals are generated from melanins, and are accelerated by UV radiation (Chedekel et al., 1980; Ye et al., 2006). In addition, our recent studies have confirmed that this type of redox reaction that produces superoxide radicals is accelerated by UVA radiation in synthetic eumelanin and pheomelanin (Ito et al., 2016; Szewczyk et al., 2016). In those studies, it was also shown that singlet oxygen is generated along with superoxide radicals from synthetic melanins. However, the generation of singlet oxygen from UVA-irradiated RD-EM and its biological implication is beyond the scope of the present study and remains to be studied. In this regard, the generation of hydroxyl radicals and singlet oxygen was suggested for the tyrosinase-catalyzed oxidation of RD and RD-catechol (Miyaji et al., 2017). The fate of the superoxide radical generated under our experimental conditions is not straightforward. Some superoxide radicals dismutate to form H2O2 and molecular oxygen. Other superoxide radicals may react with the reduced and oxidized forms of RD-EM, as has been observed with melanins (Korytowski et al., 1986; 1987). The remaining superoxide radicals are able to react


rapidly with antioxidants such as thiols (Winterboum & Metodiewa, 1999) and AA (Som et al., 1983).

Accepted Article

These scavenging actions of superoxide radicals generate H2O2. In conclusion, this study has shown that the potent pro-oxidant activity of RD-EM (Ito et al.,

2017) is enhanced by UVA radiation. These results suggest that RD induces cytotoxicity to melanocytes through tyrosinase-catalyzed activation to quinonoid products (RD-EM) that leads to the depletion of antioxidants and the generation of ROS and this process can be exacerbated by UVA radiation. In this connection, it is puzzling why RD-induced leukoderma develops in only 2-2.5% of consumers (Nishigori et al., 2015). A higher level of tyrosinase activity appears to be the most important causative factor because tyrosinase activity in cultured human melanocytes is correlated with cytotoxicity (Kasamatsu et al., 2014). An imbalance in the redox state is likely one of the causative factors, as suggested by studies on the roles of GSH and NAD(P)H quinone dehydrogenase, quinone 1 (NQO1). Thus, Kondo et al. (2016) have shown that glutathione maintenance is crucial for the survival of melanocytes after exposure to RD while Okubo et al. (2016) suggested that NQO1 attenuates the cytotoxicity of RD and/or its metabolites. Our present study suggests UVA radiation as an additional causative factor of RD-induced leukoderma, although the frequent occurrence of RD-induced leukoderma in summer months can be due to the frequent use of RD-containing cosmetics by most of users in sun-exposed area in summer. Finally, it would be interesting to study the effects of UVA radiation on RD-treated

melanocytes. Another remaining issue is the exact mechanism as to how UVA radiation increases the pro-oxidant activity of RD-EM. It would also be interesting to examine whether UVA radiation induces RD-EM to increase superoxide radical generation as compared to no radiation (Ito et al., 2017) and to generate singlet oxygen. Although the present study is not intended to examine whether RD-EM is a “phototoxic” compound (Kim et al., 2015b), it should be pointed out that the oxidation product from RD (equivalent to RD-EM) has a molar extinction coefficient of 1,000 to 2,000 from 300 nm to 600 nm (Ito et al., 2014a), which places RD-EM in a candidate for a “phototoxic” compound (Kim et al., 2015b). It would be possible that upon UVA radiation RD-EM is excited to produce radicals and generate superoxide radical and singlet oxygen.


ACKNOWLEDGEMENTS

Accepted Article

This work was supported, in part, by a Japan Health, Labour and Welfare Policy Research Grants (H29-Iyaku-Shitei-003). The authors wish to acknowledge Kanebo Cosmetics Inc. for the gift of RS-rhododendrol.

CONFLICT OF INTEREST The authors declare no conflict of interest.

REFERENCES Aoyama, Y., Ito, A., Suzuki, K., Suzuki, T., Tanemura, A., Nishigori, K., … Matsunaga, K. (2014). The first epidemiological report on rhododenol-induced leukoderma in Japan based on a nationwide survey. Japanese Journal of Dermatology, 124, 2095-2109 (in Japanese).

Chedekel, M.R., Agin, P.P., & Sayre, R.M. (1980). Photochemistry of pheomelanin – action spectrum for superoxide production. Photochemistry and Photobiololgy, 31, 553-555.

Haywood, R.M., Andrady, C., Kassouf, N., & Sheppard, N. (2011). Intensity-dependent direct solar radiation- and UVA-induced radical damage to human skin and DNA, lipids and proteins. Photochemistry and Photobiology, 87, 117-130.

Imai, Y., Ito, S., & Fujita, K. (1987). Determination of natural thiols by liquid chromatography after derivatization with 3,5-di-tert-butyl-1,2-benzoquinone. Journal of Chromatography 420, 404-410.

Ito, S., Yamashita, T., Ojika, M., & Wakamatsu, K. (2014a). Tyrosinase-catalyzed oxidation of rhododendrol produces 2-methylchromane-6,7-dione, the putative ultimate toxic metabolite: implications for melanocyte toxicity. Pigment Cell and Melanoma Research, 27, 744-753.

Ito, S., Gerwat, W., Kolbe, L., Yamashita, T., Ojika, M. & Wakamatsu, K. (2014b). Human tyrosinase is able to oxidize both enantiomers of rhododendrol. Pigment Cell and Melanoma Research, 27, 1149-1153.

Ito, S., Okura, M., Nakanishi, Y., Ojika, M., Wakamatsu, K., & Yamashita, T. (2015). Tyrosinase-catalyzed metabolism of rhododendrol (RD) in B16 melanoma cells: production of


RD-pheomelanin and covalent binding with thiol proteins. Pigment Cell and Melanoma

Accepted Article

Research, 28, 295-306. Ito, S., Kikuta, M., Koke, S., Sarna, T., & Wakamatsu, K. (2016). Mechanism of UVA-induced oxidation of 5,6-dihydroxyindole-2-carboxylic acid (DHICA) using a differential spectrophotometric method. Pigment Cell and Melanoma Research, 29, 340-351.

Ito, S., Okura, M., Wakamatsu, K., & Yamashita, T. (2017). The potent pro-oxidant activity of rhododendrol-eumelanin induces cysteine depletion in B16 melanoma cells. Pigment Cell and Melanoma Research, 30, 63-67.

Kasamatsu, S., Hachiya, A., Nakamura, S., … Matsunaga, K. (2014). Depigmentation caused by application of the active brightening material, rhododendrol, is related to tyrosinase activity at a certain threshold. Journal of Dermatological Science, 76, 16-24.

Kim, E., Panzella, L., Micillo, R., Bentley, W.E., Napolitano, A., & Payne, G.F. (2015a). Reverse engineering applied to red human hair pheomelanin reveals redox-buffering as a pro-oxidant mechanism. Scientific Reports, 5, 18447.

Kim, K., Park, H., & Lim, K.-M. (2015b). Phototoxicity: its mechanism and animal alternative test methods. Toxicology Research, 31, 97-104.

Kim, M., Baek, H.S., Lee, M., Park, H., Shin, S.S., Choi, D.W., & Lim, K.M. (2016). Rhododenol and raspberry ketone impair the normal proliferation of melanocytes through reactive oxygen species-dependent activation of GADD45. Toxicology In Vitro, 32, 339-346.

Kishida, R., Kasai, H., Aspera, S.M., Arevalo, R.L., & Nakanishi, H. (2017). Density functional theory-based first principles calculations of rhododendrol-quinone reactions: preference to thiol binding over cyclization. Journal of Physical Society of Japan, 86, 024804-1-5.

Kondo, M., Kawabata, K., Sato, K., Yamaguchi, S., Hachiya, A., Takahashi, Y., & Inoue, S. (2016). Glutathione maintenance is crucial for survival of melanocytes after exposure to rhododendrol. Pigment Cell and Melanoma Research, 29, 541-549.

Korytowski, W., Kalynaraman, B, Menon, I.A., Sarna, T., & Sealy, R.C. (1986). Reaction of superoxide anions with melanins: electron spin resonance and spin trapping studies. Biochimica et Biophysica Acta 882, 145-153.


Korytowski, W., Plas, B., Sarna, T., & Kalyanaraman, B. (1987). Photoinduced generation of

Accepted Article

hydrogen peroxide and hydroxyl radicals in melanins. Photochemistry and Photobiology, 45, 185-190.

Mitra, D., Luo, X., Morgan, A., Wang, J., Lo, J., Guerrero, C.R., … Fisher, D.E. (2012). An ultraviolet-radiation-independent pathway to melanoma carcinogenesis in the red hair/fair skin background. Nature, 491, 449-453.

Miyaji, A., Gabe, Y., Kohno, M., & Baba, T. (2017). Generation of hydroxyl radicals and singlet oxygen during oxidation of rhododendrol and rhododendrol-catechol. Journal of Clinical and Biochemical Nutrition, 60, 86-92.

Morgan, A.M., Lo, J., & Fisher, D.E. (2013). How does pheomelanin synthesis contribute to melanomagenesis?: Two distinctive mechanisms could explain the carcinogenicity of pheomelanin synthesis. Bioessays, 35, 672-675.

Nagata, T., Ito, S., Itoga, K., Kanazawa, H., & Masaki, H. (2015). The mechanism of melanocyte-specific cytotoxicity induced by phenol compounds having a prooxidant effect, relating to the appearance of leukoderma. BioMedical Research International, 2015, 479798.

Napolitano, A., Panzella, L., Monfrecola, G., & d’Ischia, M. (2014). Pheomelanin-induced oxidative stress: bright and dark chemistry bridging red hair phenotype and melanoma. Pigment Cell and Melanoma Research, 27, 721-733.

Nishigori, C., Aoyama, Y., Ito, A., Suzuki, K., Suzuki, T., Tanemura, A., … Matsunaga, K. (2015). Guide for medical professionals (i.e. dermatologists) for the management of rhododenol-induced leukoderma. Journal of Dermatology, 42, 113-128.

Okubo, A., Yasuhira, S., Shibazaki, M., Takahashi, K., Akasaka, T., Masuda, T., & Maesawas, C. (2016). NAD(P)H dehydrogenase, quinone 1 (NQO1), protects melanin-producing cells from cytotoxicity of rhododendrol. Pigment Cell and Melanoma Research, 29, 309-316.

Okura, M., Yamashita, T., Ishii-Osai, Y., Yoshikawa, M., Sumikawa, Y., Wakamatsu, K., & Ito, S. (2015). Effects of rhododendrol and its metabolic products on melanocytic cell growth. Journal of Dermatological Science, 80, 142-149.

Panzella, L., Leone, L., Greco, G., Vitiello, G., D’Errico, G., Napolitano, A., & d’Ischia, M. (2014). Red human hair pheomelanin is a potent pro-oxidant mediating UV-independent contributory mechanisms of melanomagenesis. Pigment Cell and Melanoma Research, 27, 244-252.


Sasaki, M., Kondo, M., Sato, K., Umeda, M., Kawabata, K., Takahashi, Y., … Inoue, S. (2014).

Accepted Article

Rhododendrol, a depigmentation-inducing phenolic compound, exerts melanocyte cytotoxicity via a tyrosinase-dependent mechanism. Pigment Cell and Melanoma Research, 27, 754-763.

Som, S., Raha, C., & Chatterjee, B. (1983). Ascorbic acid: a scavenger of superoxide radical. Acta Vitaminology & Enzymology, 5, 243-250.

Szewczyk, G., Zadlo, A., Sarna, M., Ito, S., Wakamatsu, K., & Sarna, T. (2016). Aerobic photoreactivity of synthetic eumelanins and pheomelanins: generation of singlet oxygen and superoxide anion. Pigment Cell and Melanoma Research, 29, 669-678.

Winterboum, C.C., & Metodiewa, D. (1999). Reactivity of biologically important thiol compounds with superoxide and hydrogen peroxide. Free Radical in Biology and Medicine, 27, 322-328.

Ye, T., Hong, L., Garguilo, J., Pawlak, A., Edwards, G.S., Nemanichi, R.J., … Simon, J.D. (2006). Photoionization thresholds of melanin obtained from free electron laser – photoelectron emission microscopy, femtosecond transient absorption spectroscopy and electron paramagnetic resonance measurements of oxygen photoconsumption. Photochemistry and Photobiology 82, 733-737.

Zhou, M., Diwu, Z., Panchuk-Voloshina, N., & Haugland, R.P. (1997). A stable nonfluorescent derivative of resorufin for the fluorometric determination of trace hydrogen peroxide: applications in detecting the activity of phagocyte NADPH oxidase and other oxidases. Analytical Biochemistry, 253, 162-168.

Figure Legends Figure 1. Tyrosinase-catalyzed oxidation of rhododendrol (RD, 1) giving rise to RD-EM. Our previous study (Ito et al., 2014a) showed that the immediate product is RD-quinone (2) which undergoes addition of water molecule and is oxidized to form RD-hydroxy-p-quinone (3). RD-hydroxy-p-quinone gradually dimerizes to form RD-EM. We originally proposed that RD-cyclic quinone is produced through cyclization (Ito et al., 2014a). However, a recent theoretical study by Kishida et al. (2017) indicated that this cyclization does not proceed fast enough. It would be more appropriate to consider that RD-cyclic quinone (+ water molecule) is produced in an equilibrium with RD-hydroxy-p-quinone as described in Ito et al. (2014a). The oxidized form of RD-EM is able to oxidize thiols (R-SH), ascorbic acid (AA) and NADH to disulfides (RS-SR), dehydroascorbic acid (DeAA) and NAD+, respectively, while the reduced form of RD-EM is able to reduce molecular


oxygen producing superoxide radicals. It is likely that RD-EM is a mixture of dimers and tetramers

Accepted Article

(Ito et al., 2017). The present study shows that the pro-oxidant activity of RD-EM is enhanced by UVA radiation.

Figure 2. Time course of oxidation of the antioxidants by RD-EM with or without UVA radiation. (A) Changes in content of GSH. (B) Changes in content of CySH. (C) Changes in content of AA. (D) Changes in content of NADH. Each oxidation was performed with 910 M antioxidant and 91 M RD-EM at pH 7.4 Experiments were repeated two or three times, each performed in duplicate. Values reported are means ± SEM of two (AA and NADH) or three (GSH and CySH) experiments (four or six data points).

Figure 3. Roles of oxygen in the pro-oxidant activity of RD-EM. (A) Effect of oxygen. The decreases of CySH (910 M) by RD-EM (91 M) after 120 min reaction with or without UVA radiation are compared with or without oxygen. The experiment with oxygen was carried out under air while that without oxygen was under an argon atmosphere by purging with an argon stream for 2 min. Values reported are means ± SEM of two experiments (four data points). Differences between the presence and the absence of oxygen are statistically significant at P < 0.0001. (B) Production of H2O2. The production of H2O2 from antioxidants (910 M) incubated with RD-EM (91 M) for 120 min were compared with or without UVA radiation. Values reported are means ± SEM of two or three experiments (four to six data points). Difference in NADH oxidation is statistically significant at P < 0.0001. (C) Consumption of H2O2. The consumption of H2O2 (910 M) by antioxidants (455 M) incubated for 60 min at 30˚C was compared. Blank experiments with buffer and with tyrosinase (50 U) alone consumed H2O2 by 2% and 22%. Values reported are means ± SEM of three experiments (three data points).


Table 1. Comparison of the rate of oxidation of antioxidants by RD-EM with or without UVA

Accepted Article

radiation1

1

GSH

CySH

AA

NADH

Kinetics2

Zero-order

Zero-order

First-order

First-order

No UV

0.84 ± 0.15

4.29 ± 0.05

4.07 ± 0.58

0.84 ± 0.07

UVA

3.55 ± 0.25

8.42 ± 0.35

9.95 ± 0.24

3.27 ± 0.10

UVA/No UV

4.2

2.0

2.4

3.9

No UV, Control3

Not determined4

0.87 ± 0.11

0.34 ± 0.10

0.15 ± 0.08

UVA, Control3

Not determined4

1.35 ± 0.21

0.53 ± 0.12

1.49 ± 0.08

Rates are expressed as M/min. For the first-order kinetics, the initial rates are presented. Values

reported are means ± SEM of four (AA and NADH) or six (GSH and CySH) data points. Differences between No UV and UVA are statistically significant at P < 0.001. 2

The kinetics of the oxidations was compared between the zero-order and the first-order kinetics (see

Figure S1). The kinetics that gave greater R2 values was selected. 3

These experiments were performed in the absence of RE-EM (but tyrosinase added).

4

These experiments were not performed because GSH was not oxidized under similar experimental

conditions (Ito et al., 2017).


SUPPORTING INFORMATION

Accepted Article

Additional supporting information may be found in the online in the supporting information tab for this article


Accepted Article This article is protected by copyright. All rights reserved.

Accepted Article This article is protected by copyright. All rights reserved.