J. Cosmet. Sci., 56, 17-27 Oanuary/February 2005) L-Ergothioneine scavenges superoxide and singlet oxygen and suppresses TNF-a and MMP-1 expression in UV-irradiated human dermal fibroblasts KEI OBAYASHI, KOUJI KURIHARA, YURI OKANO, HITOSHI MASAKI, and DANIEL B. YAROSH, Cosmos Technical Center Co. Ltd, 3-24-3, Hasune, Itabashi-ku, Tokyo, 174-0046Japan (K.0., K.K., Y.O., H.!VI.), and AGI Dermatics, 205 Buffalo Avemte, Freeport, NY 11520-4709 (D.B. Y.). Accepted for publication November 16, 2004. Synopsis Ergothioneine (EGT) is a sulfur-containing amino acid, and is presumed to function as a natural antioxidant. The purpose of this study was co identify the nature of the antioxidant activity and investigate the effects of EGT on UV-induced cellular response. In chemical studies, EGT scavenged the superoxide anion radical (•02-) and singlet oxygen ( 1 02). In cultured fibroblasts, EGT suppressed TNF-a up-regulation by UVB irradiation. In addition, in fibroblasts exposed to UV-A, EGT suppressed the expression of matrix metal­ loproteinase 1 (MMP-1) protein by nearly 50% and reduced MMP-1 mRNA expression. From these results, we conclude that EGT scavenges reactive oxygen species generated by both Type I and Type II photosen­ sitization and suppresses both TNF-a expression and MMP-1 at their transcriptional level. EGT may reduce skin anti-aging effects after UV irradiation by the scavenging of •02 - and 1 02, and reducing signals for protease and inflammatory activity. INTRODUCTION Recently, the increase in the aged population and the increase in UV at the earth's surface (1) have focused the public's concern on the long-term effects of UVA (320 nm-400 nm) and UVB (290 nm-320 nm), especially the acceleration of premature skin aging. Photoaged facial skin is characterized by the appearance of deep wrinkles at the corner of the eyes and around the mouth. Many studies have demonstrated that the alterations of the extracellular matrix at the papillary dermis, collagen, and elastin substantially contributes to the formation of photoaged skin (2-8). The decrease of collagen fibers and the disappearance of elastin fine fiber and oxytalan fiber has been observed in photoaged skin. These alterations are caused by repeated UV exposure. Address all correspondence to Hitoski Masaki. 17

18 JOURNAL OF COSMETIC SCIENCE UV A exposure to dermal fibroblasts leads to the reduction of collagen synthesis (8) and the excess elevation of matrix metalloproteinase-1 (MMP-1)/interstitial collagenase (9). MMP-1 is a member of the MMPs, a superfamily of endopeptidase that is capable of degrading extracellular matrix components (10). Excess expression of MMP-1 by skin fibroblasts causes subsequent damage of dermal connective tissue. The imbalance be­ tween the synthesis and degradation of collagen critically contributes to the process of matrix alteration (11) and leads to photoaging. UVB causes acute damage in the skin, such as DNA damage and apoptosis of keratino­ cytes, even in dermal cells (12). In addition, UVB induces the production of cytokines, hormones and chemical messengers, IL-1, TNF-a, propiomelanocortin-derived hor­ mones, and prostaglandin E2, which consequently leads to erythema and inflammation in the dermis (13). UVB radiation creates superoxide anion (0 2 -) (type I photosenitization) due to reaction with water, activation of mitochondrial function, and release of peroxides by inflamma­ tory cells (14), while UV A radiation generates singlet oxygen (10 2 ) (type II photosen­ sitization) through photosensitization reactions with several intracellular chromophors, such as NADH, NADPH, and flavine protein (15). It has been reported that 1 0 2 generated by UV A mediates the induction of MMP-1 through the pathway of IL-6 and IL-1 (16,17). Furthermore, a much higher level of oxidative protein was observed at the papillary dermis of photoaged skin compared to younger skin (18). This evidence indicates that the process of skin photoaging is in part mediated by oxidative stress, including reactive oxygen species (ROS). Therefore, an antioxidant possessing a wide spectrum of ROS scavenging should prevent UV-induced skin damage. Ergothioneine (EGT) is a natural antioxidant, and an amino acid not incorporated into protein, whose sulfur is predominantly in the thione form (Figure 1). EGT is a fungal metabolite that cannot be endogenously synthesized by mammals and must be taken up in the diet (19). It is found in many mammalian tissues in millimolar quantities (19). EGT is generally regarded as an antioxidant, although results are conflicting. Some regard it as a scavenger of hydrogen peroxide (20), while others contend that it does not readily react with hydrogen peroxide but does scavenge hydroxyl radicals (21). Also, some data indicate that EGT quenched 1 02 by monitoring 1270-nm phosphorescence derived from 1 02 (22). In this study, we examined the scavenging abilities of EGT against •02 - and 1 0 2 using chemical and biological systems to identify antioxidative characters. In addition, the effects of EGT on UV-induced cellular responses such as expression of both TNF-a and MMP-1 were evaluated. Figure l. Chemical structure of L-ergothioneine (EGT).