SOD1 was provided by Nutrex Technology Corporation (Seoul, Korea). Mushroom tyrosinase, α-MSH, _L-DOPA, and phenylthiourea were purchased from Sigma-Aldrich Co. (St. Louis, MO, USA). Dulbecco's modified Eagle's medium (DMEM), trypsin-ethylenediaminetetraacetic acid, phosphate-buffered saline, and penicillin/streptomycin were purchased from WelGENE Biopharmaceuticals (Daegu, Korea). Fetal bovine serum (FBS) was purchased from Life Technologies (Gibco; Life Technologies, Grand Island, NY, USA).

B16F10 murine melanoma cells (CRL-6475) were purchased from the American Type Culture Collection (Rockville, MD, USA). Cells were maintained in DMEM supplemented with 10% FBS and 1% penicillin-streptomycin in 5% CO₂ at 37℃. Cells were passaged every 3 days until a maximum passage number of 20 was achieved.

Male, 4-week-old, HRM-2 melanin-possessing hairless mice (total=9) were obtained from Hoshino Laboratory Animals (Saitama, Japan) and housed in an air-conditioned room maintained at 24℃±2℃ and 55%±15% humidity with a 12-hours light-dark cycle. The mice were placed individually in separate cages and fed ad libitum. All procedures involving animals were conducted in accordance with the guidelines of the Institutional Animal Care and Use Committee of Chung-Ang University in Korea (IRB Number: 13-0013). After a 1-week stabilization period, mice were randomly divided into three groups of three animals each. The mice were anesthetized with Zoletil 50 (50 mg/kg) and xylazine (10 mg/kg). SOD1 was dissolved in a vehicle consisting of ethanol and propylene glycol at a ratio of 3:7, and then topically applied to a designated site on the dorsal skin of the back of the animal once a day. Mice were exposed to UVB using an NB-UVB lamp (Dermalight Psoracomb UV-B-311nm-narrowband; National Biological Corp., Twinsburg, OH, USA) and a UV-B Narrowband PL-L/PL-S lamp (Philips, Eindhoven, Netherlands) at 190 mJ/cm². The experiment schedule is shown in Table 124,25. The color of the skin sites was measured using a spectrophotometer (CR-10; Konica Minolta Sensing Inc., Osaka, Japan), and the colors are described by L-values according to the Commission Internationale de l'Eclairage color system. The mice were killed and their skin was quickly removed and immediately frozen in liquid nitrogen and stored at -80℃.

B16F10 melanoma cells were seeded in 96-well plates (2×10⁴ cells/well). After 24 hours incubation at 37℃, the medium was replaced with media containing SOD1 diluted to the appropriate concentration. Control cells were treated with distilled water (DW) at a final concentration of 0.1%. After 24 hours, the media containing the compounds or DW were replaced with media containing 10% EZ-CyTox (Daeil Lab Service, Seoul, Korea). The cells were then incubated at 37℃ for 30 minutes, and the absorbance was measured using an enzyme-linked immunosorbent assay (ELISA) reader (VERSAMax; Molecular Devices, Sunnyvale, CA, USA) at a wavelength of 450 nm. All assays were performed in triplicate. The cytotoxic effect of each treatment was expressed as a percentage of cell viability relative to the untreated control cells.

Tyrosinase activity was assayed as a function of DOPA oxidase activity. A total of 70 µl of 0.1 M phosphate buffer (pH 6.8) containing SOD1 was mixed with 20 µl of 10 µg/ml mushroom tyrosinase in a well of a 96-well plate, and 10 µl of 10 mM _L-DOPA was added. Absorbance values were measured every 10 minutes for at least 1 hour at 475 nm using an ELISA reader at an incubation temperature of 37℃. The quantity of dopachrome formed in the reaction mixture was determined against a blank (solution without enzyme) at 475 nm in the ELISA reader. The percentage of tyrosinase activity was calculated as follows: tyrosinase activity (%)=[(A-B)/(C-D)]×100, where A is the absorbance of the reaction mixture containing the test sample and mushroom tyrosinase, B is the absorbance of the blank sample containing the test sample but no mushroom tyrosinase, C is the absorbance of the reaction mixture without the test sample and with mushroom tyrosinase, and D is the absorbance of the well lacking both the test sample and mushroom tyrosinase (_L-DOPA alone).

B16F10 melanoma cells were incubated at a density of 1×10⁵ cells in 6-well plates overnight. The α-MSH (1 µm) was then added, and the cells were treated with increasing concentrations of substance in phenol red-free DMEM for 3 days. Two hundred-microliter aliquots of medium were then placed in 96-well plates, and the optical density of each culture well was measured using an ELISA reader at 405 nm. The data were normalized to the protein content of the cell lysates. The cell lysates were subsequently processed for determining the protein concentration using a BCA Protein Assay Kit (Pierce Biotechnology, Rockford, IL, USA). Melanin production was expressed as the percentage of living cells compared to the controls.

Skin samples were fixed in 4% paraformaldehyde overnight at room temperature and stained for melanin using a Fontana-Masson staining kit (American Mastertech Inc., Lodi, CA, USA) according to the manufacturer's instructions. Briefly, sliced skin samples were stained with ammoniacal silver solution for 60 minutes at 60℃, followed by incubation in 0.1% gold chloride and then in 5% sodium thiosulfate. The tissue was also stained with hematoxylin and eosin for morphological analyses. Staining was examined under a phase-contrast microscope (Eclipse TS100; Nikon Instruments Inc., Melville, NY, USA).

Data are presented as mean±standard deviation, and statistical comparisons between treated groups and the untreated group were performed using one-way ANOVA followed by post hoc Tukey tests for direct comparison between specific groups (PASW Statistics ver. 18.0; IBM Co., Armonk, NY, USA). p<0.05, p<0.01, and p<0.001 were considered statistically significant.

To determine if SOD1 could inhibit melanogenesis, B16F10 melanoma cells were treated with the SOD1 before α-MSH stimulation. We first tested the cytotoxicity of SOD1 in B16F10 melanoma cells using the EZ-CyTox assay. B16F10 melanoma cells were treated with SOD1 at concentrations of 1 to 1,000 ng/ml for 24 hours. SOD1 was found to have no cytotoxic effect on B16F10 melanoma cells at the concentrations used (Fig. 1).

To investigate the direct effects of SOD1 on tyrosinase activity, we measured the tyrosinase activities of mushroom tyrosinase in a cell-free system, as described in the Materials and Methods. However, SOD1 was found to have no direct inhibitory effect on mushroom tyrosinase activity at concentrations ranging from 1 to 1,000 ng/ml (Fig. 2).

The effect of SOD1 on the melanogenesis of B16F10 melanoma cells was examined. To determine whether SOD1 inhibits α-MSH-induced melanin synthesis in B16F10 melanoma cells, we measured the α-MSH-induced melanin production of B16F10 melanoma cells after 3 days of SOD1 treatment at 10 to 1,000 ng/ml. SOD1 considerably inhibited melanin synthesis when used at concentrations greater than 10 ng/ml (Fig. 3). SOD1 treatment also reduced α-MSH-induced melanin release in a dose-dependent manner.

The effect of SOD1 on skin pigmentation was examined in vivo in HRM-2 melanin-possessing hairless mice treated according to the schedule shown in Table 1. Treatment of animals with SOD1 dissolved in vehicle did not cause any noticeable skin irritation. Exposure of mice to UVB led to decreased ΔL (lightness) values, which represented a pigmentation effect. The UVB-induced changes in ΔL (lightness) values were significantly reversed in SOD1-treated animals, and the color properties of the skin of SOD1-treated animals were comparable to those of the control groups (Fig. 4). The pigmentation pattern was photographed using a Dermoscope (DermLite Pro, California, USA) and a Folliscope (LeedM, Seoul, Korea) (Fig. 4). In addition, SOD1 was found to have a protective effect on UVB-induced epidermal and dermal changes (Fig. 5). To visualize the melanin, the skin samples were stained with Fontana-Masson stain and examined. As shown in Fig. 6, the amount of melanin granules was significantly increased by UVB irradiation (arrows in Fig. 6B). Topical application of SOD1 inhibited the UVB-induced increase in melanin generation (Fig. 6C).