Superoxide dismutase 1 inhibits alpha-melanocyte stimulating hormone and ultraviolet B-induced melanogenesis in murine skin

Abstract

Background: Over the last decade, the incidence of ultraviolet B (UVB)-related skin problems has increased. Oxidative stress caused by UVB induces the secretion of melanocyte growth and activating factors from keratinocytes, which results in the formation of cutaneous hyperpigmentation. Therefore, increasing the antioxidant abilities of skin cells is thought to be a beneficial strategy for the development of sunscreen agents. Superoxide dismutase 1 (SOD1) is an antioxidant enzyme that is known to exhibit antioxidant properties.

Objective: The purpose of this study was to investigate the effect of SOD1 on alpha-melanocyte stimulating hormone (α-MSH) and UVB-induced melanogenesis in B16F10 melanoma cells and HRM-2 melanin-possessing hairless mice.

Methods: The inhibitory effect of SOD1 on tyrosinase activity was evaluated in a cell-free system. Additional experiments were performed using B16F10 melanoma cells to demonstrate the effects of SOD1 in vitro, and HRM-2 melanin-possessing hairless mice were used to evaluate the antimelanogenic effects of SOD1 in vivo.

Results: We found that SOD1 inhibited melanin production in a dose-dependent manner without causing cytotoxicity in B16F10 melanoma cells. SOD1 did not inhibit tyrosinase activity under cell-free conditions. The results indicate that SOD1 may reduce pigmentation by an indirect, nonenzymatic mechanism. We also found that SOD1 decreased UVB-induced melanogenesis in HRM-2 melanin-possessing hairless mice, as visualized through hematoxylin and eosin staining and Fontana-Masson staining.

Conclusion: Our results indicate that SOD1 has an inhibitory effect on α-MSH and UVB-induced melanogenesis, indicating that SOD1 may be a promising sunscreen agent.

Keywords: Alpha-MSH; HRM-2; Skin pigmentation; Superoxide dismutase 1; Ultraviolet radiation.

Fig. 1

Cell viability of B16F10 melanoma cells treated with superoxide dismutase 1 (SOD1) at different concentrations. Cells were treated with SOD1 at 1 to 1,000 ng/ml for 24 hours. Cell viability was determined using EZ-CyTox assays. Results are expressed as the percent viability relative to controls. Most of the B16F10 melanoma cells were viable at concentrations in the range of 1 to 1,000 ng/ml. Each measurement was made in triplicate and data represent the mean±standard deviation.

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.

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.

Fig. 4

Effect of superoxide dismutase 1 (SOD1) on the level of ultraviolet B (UVB)-induced melanogenesis in HRM-2 melanin-possessing hairless mice. HRM-2 melanin-possessing hairless mice were pretreated with vehicle or SOD1 on a designated site on the dorsal skin and then UVB-irradiated according to the indicated schedule. The picture represents the dorsal skin of the HRM-2 melanin-possessing hairless mice after treatment. For statistical analysis, ΔL (lightness) values of each animal were calculated as the average values after UVB exposure (day 17) minus the average baseline values before any treatment (day 0). Values represent the mean±standard deviation of five experiments. For statistical analysis, results of one-way ANOVA were used: ^*p<0.05, as compared with UVB-treated controls. (A) ΔL (lightness) values. (B) D3200. (C) Dermoscope. (D) Folliscope.

Fig. 5

Effect of superoxide dismutase 1 (SOD1) on ultraviolet B (UVB) irradiation-induced histopathological changes in skin tissue. Change in histopathological features of the skin after repeated treatment with SOD1. Representative histopathological images of UVB-irradiated skin lesions stained by H&E (×200). (A) Control. (B) UVB 190 mJ/cm². (C) 1,000 ng/ml SOD1+UVB 190 mJ/cm².

Fig. 6

Effect of superoxide dismutase 1 (SOD1) on ultraviolet B (UVB) irradiation-induced hyperpigmentation changes in skin tissue. Change in histopathological features of the skin after repeated treatment with SOD1. Representative histopathological images of UVB-induced skin pigmentation stained by Fontana and Masson (×200). Melanin pigments (arrows) are stained black. (A) Control. (B) UVB 190 mJ/cm². (C) 1,000 ng/ml SOD1+UVB 190 mJ/cm².