Cell culture

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.

Animals and skin color determination

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 1^24,25. 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.

Table 1

The experimental schedule of UVB irradiation in HRM-2 melanin-possessing hairless mice

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SOD1: superoxide dismutase 1, UVB: ultraviolet B.

Cell viability assay

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.

Cell-free enzymatic assay for tyrosinase activity

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).

Measurement of melanin content

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.

Histological analysis

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).

Effects of superoxide dismutase 1 on tyrosinase activity in B16F10 melanoma cells

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).

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Fig. 2

Inhibitory effects of superoxide dismutase 1 (SOD1) on tyrosinase activity in cell-free assays. Mushroom tyrosinase was treated with SOD1 at 1 to 1,000 ng/ml. SOD1 did not appear to inhibit mushroom tyrosinase activity. Each measurement was made in triplicate and data shown represent the mean±standard deviation.

Effect of superoxide dismutase 1 on melanogenesis in B16F10 melanoma cells

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.

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Fig. 3

Effect of superoxide dismutase 1 (SOD1) on melanin content in B16F10 melanoma cells. SOD1 was tested at 10, 100, and 1,000 ng/ml in B16F10 melanoma cells. B16F10 melanoma cells were co-cultured with alpha-melanocyte stimulating hormone (α-MSH) (1 µm) and SOD1 at 10 to 1,000 ng/ml for 72 hours, and compared with untreated and α-MSH-stimulated control cells. Levels of inhibition of melanogenesis are expressed as percentages of the control. The results are averages of three independent experiments and the data are expressed as mean±standard deviation. Inhibition of melanin content was related to exposure to SOD1 in a dose-dependent manner. ^**p<0.05, ^***p<0.001 as compared with α-MSH-treated controls.

This research was financially supported by the Ministry of Knowledge Economy (MKE), Korea Institute for Advancement of Technology (KIAT) through the Inter-ER Cooperation Projects.