Purpose: Products of ultraviolet (UV) irradiation such as reactive oxygen species (ROS) and nitric oxide (NO) cause cell death. To protect the skin from UV damage, melanin, a dark colored pigment, is produced by melanocytes. While melanin is essential for skin health, a demand for melanin scavenging preparations exists. To protect from ROS and NO, thiol compounds with anti-oxidant effect has been widely used. However, the skin whitening effect of thiols is very weak, a demand for more effective ROS and NO scavenging agents exists. Therefore, we focused on reactive sulfur species (RSS). RSS including cysteine persulfide have recently been reported to have stronger anti-oxidant effects than thiols. Sodium polysulfides (Na2Sn), diallyltrisulfide (DATS) and dimethyltrisulfides (DMTS) are commonly used as RSS donors. However, the half-lives of these compounds are very short and have offensive smells. Thus, the development of novel RSS- delivery-systems would be highly desirable. Human serum albumin (HSA) is the most abundant protein in serum and is widely used as a drug carrier because of its biocompatibility and long plasma retention properties. HSA contains a total of 35 Cys residues and one of them, Cys34, is present in the form of a free thiol group. Consequently, we hypothesized that HSA could be used as a RSS carrier via the S-sulfhydration of Cys34-SH. Ogasawara et al. previously prepared sulfur-bound serum albumin reacted with sodium sulfide (NaHS) by a simple mixing of the reagents. Hence, we developed the preparation of Na2Sn-treated HSA and evaluated its use as a skin brightening agent.
Methods: 【Synthesis of Sn-Alb】
Alb (300 mM) was incubated with 1 mM of sodium polysulfides (Na2Sn) in PBS (pH 7.4) for 1 h at 37oC. After the reaction, excess sodium polysulfides were removed by gel filtration with a Sephadex G-25 column.
【Determination of sulfur binding rate by probe】
Sample (20 mM) was incubated with of SSP4 (5 mM) in 1 mM Cetyltrimethylammonium Bromide / PBS (pH 7.4) for 10 min at 25℃. After incubation, the fluorescence measured at 457 nm, emission at 490-535 nm.
【Elimination Method of Sulfide from Polysulfide (EMSP)】
Samples were incubated with 1 M L-ascorbic acid and 3 M potassium hydroxide for 4 h at 37℃. Level of eliminated sulfide from sulfane sulfur was determined by methylene blue assay.
B16 melanoma cells were seeded in 24 well plates at a concentration of 2.5×104 cells/well and cultured under 5% CO2 at 37oC for 24 h. Samples were treated with 0.4 mM tyrosine and 10 mM NH4Cl in DMEM containing 10% FBS and then incubated under 5% CO2 at 37oC for 72 h. After the incubation, the cells were washed twice with PBS and dissolved in 1 N NaOH (200 mL). After a 2 h incubation on 60oC, the absorption (405 nm) was measured by means of a micro-plate reader.
Results: Based on the EMSP analyses, the level of S-sulfydration increased independently of the amount of sulfur. On the other hand, the Na2S3- or Na2S4-treatment enhanced the SSP4 fluorescence intensity compared with the Na2S- or Na2S2-treatments, suggesting that SSP4 possibly reacted with the polysulfide of the protein in a non-linear manner. DPPH radical showed that the Na2Sn-treated HSA had a significantly higher concentration of added sulfur. It was also showed that Na2Sn-treated HSA inhibited melanin synthesis and the inhibition was dependent on the sulfur content. Cell images of B16 melanoma cells after the application of the Na2Sn-treated HSA also demonstrated that Na2Sn-treated HSA decreased the ratio of production of melanin positive cells. The fluorescence probes indicate that the Na2S4-treated HSA caused a significant decrease in the fluorescence of CMH2-DCF-DA to PBS and HSA by irradiation at 254 nm and 365 nm. Some commercial anti-melanin agents are known to directly inhibit tyrosinase activity. Thus, we tested whether the Na2Sn-treated HSA altered the activity of tyrosinase. The findings indicated that the Na2Sn- treated HSA inhibited mushroom tyrosinase to a greater extent than non-treated HSA. Finally, skin irritation tests for the Na2S4-treated HSA was performed using 3D cultured human skin cells according to OECD guidelines. As a result, the numbers of surviving cells were not decreased by Na2S4-treated HSA with/without the use of a topical cream.
Conclusion: In conclusion, we reported on the development of a novel RSS de- livery system using serum albumin as a stable carrier. Reactive sulfur, when combined with HSA, had a stronger anti-oxidant effect than HSA and inhibited melanin synthesis in melanoma cells. The mechanism of anti-melanogenesis involves not only ROS and NO scavenging, but also suppression of tyrosinase activity and melanin aggregation. Hence, Na2Sn-treated HSA has considerable potential for use as a safe skin whitening agent.
Yu Ishima– Associate professor, Tokushima University
Hiroshi Watanabe– Associate professor, Kumamoto University, Kumamoto
Toru Maruyama– Professor, Kumamoto University, Kumamoto
Tatushiro Ishida– Professor, Tokushima University