Squalene as a target molecule in skin hyperpigmentation caused by singlet oxygen.

Based on our previous finding (Biochem. Biophys. Res. Commun., 223, 578-582, 1996) of singlet oxygen generation from coproporphyrin excreted on the skin surface from Propionibacterium acnes, we hypothesized that singlet oxygen formed in this way under UV exposure would promote peroxidation of skin surface lipids. We found that squalene was oxidized efficiently by singlet oxygen derived from coproporphyrin under UV exposure, and that the rate constant of squalene peroxidation by singlet oxygen was ten-fold higher than that of other skin surface lipids examined. The reaction was promoted more efficiently by UVA than by UVB. Furthermore, we found that topical application of squalene peroxide induced skin hyperpigmentation through increasing prostaglandin E(2) release from keratinocytes in guinea pigs. These results suggest that squalene peroxide formation by singlet oxygen plays a key role in photo-induced skin damage.

Increased levels of a particular phosphatidylcholine species in senescent human dermal fibroblasts in vitro.

Plasma membranes are essential components of living cells, and phospholipids are major components of cellular membranes. Here, we used liquid chromatography/mass spectrometry to investigate changes in the membrane phospholipid content that occur in association with aging. Our results indicate that the levels of a particular species of phosphatidylcholine comprised of stearic acid and arachidonic acid increased with age. To determine the reason for the increased levels of this particular phosphatidylcholine, we examined the effect of highly unsaturated fatty acids, such as arachidonic acid and eicosapentaenoic acid, on cellular aging. Applied arachidonic acid was incorporated into phosphatidylcholine molecules, but neither arachidonic acid nor other related unsaturated fatty acids had any effect. We conclude that increased levels of this distinctive phosphatidylcholine are a result of in vitro senescence.

Topical application of plant extracts containing xanthine derivatives can prevent UV-induced wrinkle formation in hairless mice.

BACKGROUND: Plants are the source of important products with nutritional and therapeutic value. Topical or oral administration of some plant extracts has been shown to reduce photodamage. Cacao bean and cola nut are popular edible plants that contain polyphenols and xanthine derivatives. These plant extracts possess protective effects against UV-induced erythema when taken orally, and an H(2)O(2)-scavenging effect. METHODS: Plant extracts containing xanthine derivatives and three xanthine derivatives were topically applied to the dorsal skin of hairless mice, and the mice were exposed to a resemblance of solar ultraviolet irradiation at a dose of 13.0 J/cm(2) (UVA) for 15 weeks, five times a week on weekdays. After the final irradiation, histological, and analytical studies were performed. RESULTS: Topical application of plant extracts (cacao beans, cola nuts) and caffeine, theobromine, and theophylline markedly prevented photodamage including wrinkle formation and histological alterations. A significant increase in total hydroxyproline content caused by UV irradiation was observed. In contrast, topical application of plant extracts and xanthine derivatives reduced total hydroxyproline and pepsin-resistant hydroxyproline content in comparison with that of the control (vehicle, UV-irradiation group). Moreover, naphthol AS-D chloroacetate esterase staining and diaminobenzidine staining suggested that leukocytes including neutrophils increased in the UV-exposed skin. In contrast, weak staining was observed in skin treated with xanthine derivatives. CONCLUSION: Topical application of plant extracts and xanthine derivatives suppressed wrinkle formation, dermal connective alteration, and collagen accumulation. It is suggested that xanthine derivatives prevented neutrophil infiltration caused by UV-irradiation.

Protective effects of an antioxidant derived from serine and vitamin B6 on skin photoaging in hairless mice.

The generation of reactive oxygen species (ROS) by ultraviolet radiation (UVR) accelerates skin aging, which is known as photoaging. Because cutaneous iron catalyzes ROS generation, sequestering iron by chelating agents is thought to be an effective approach toward preventing photoaging. Previously, N-(4-pyridoxylmethylene)-l-serine (PYSer) was designed as an antioxidant to suppress iron-catalyzed ROS generation by its iron-sequestering activity. In this study, PYSer showed protective effects against skin damage in hairless mice irradiated with ultraviolet B (UV-B). Topical application of PYSer to the skin significantly delayed and/or decreased the visible wrinkle formation induced by chronic UV-B irradiation. A histological study indicated that UV-B-induced epidermal hypertrophy and lymphocytic infiltration were suppressed by PYSer. Moreover, PYSer showed suppressive activity against the UV-B-induced increase in glycosaminoglycans (GAG). These results indicate that PYSer is a promising antioxidant for the prevention of chronic skin photoaging by its iron-sequestering activity.

Ergocalciferol promotes in vivo differentiation of keratinocytes and reduces photodamage caused by ultraviolet irradiation in hairless mice.

BACKGROUND: Ergocalciferol (VD(2)) is usually administered orally and it is metabolized to produce its biologically active metabolites in the liver and kidney. Active vitamin D is a well-known potent regulator of cell growth and differentiation. PURPOSE: Active vitamin D such as 1,25-dihydroxyvitamin D(3) (1alpha,25(OH)(2)D(3)) prevents photodamage, including wrinkles and morphologic alterations. However, its clinical and cosmetic use is limited because of its potent, associated effect on calcium metabolism. We examined the efficacy of vitamin D analogues with few adverse effects for preventing skin photodamage. METHOD: Topical application of VD(2) to hairless mouse dorsal skin, and exposure to solar-simulating ultraviolet (UV) radiation at a dose of 10.8 J/cm(2) (UVA) were performed for 15 weeks, five times a week on weekdays. At the end of the final irradiation, histological and analytical studies were performed. RESULTS: Topical application of VD(2) significantly prevented wrinkle formation and abnormal accumulation of extracellular matrix components. In addition, VD(2) suppressed excessive secretion of IL-6 induced by UV irradiation in cultured human normal keratinocytes, in a dose-dependent manner. CONCLUSION: VD(2) promoted keratinocytes differentiation in the epidermis and showed diverse physiological effects, the same as the active form of VD(3). The results suggested that the suppression of skin photodamage involved the promotion of keratinocytes differentiation and suppression of IL-6 secretion induced by exposure to UV. Topical application of VD(2) may become an effective means to suppress solar UV-induced human skin damage.

Active oxygen species generated from photoexcited fullerene (C60) as potential medicines: O2-* versus 1O2.

To characterize fullerenes (C(60) and C(70)) as photosensitizers in biological systems, the generation of active oxygen species, through energy transfer (singlet oxygen (1)O(2)) and electron transfer (reduced active oxygen radicals such as superoxide anion radical O(2)(-)* and hydroxyl radical *OH), was studied by a combination of methods, including biochemical (DNA-cleavage assay in the presence of various scavengers of active oxygen species), physicochemical (EPR radical trapping and near-infrared spectrometry), and chemical methods (nitro blue tetrazolium (NBT) method). Whereas (1)O(2) was generated effectively by photoexcited C(60) in nonpolar solvents such as benzene and benzonitrile, we found that O(2)(-)* and *OH were produced instead of (1)O(2) in polar solvents such as water, especially in the presence of a physiological concentration of reductants including NADH. The above results, together with those of a DNA cleavage assay in the presence of various scavengers of specific active oxygen species, indicate that the active oxygen species primarily responsible for photoinduced DNA cleavage by C(60) under physiological conditions are reduced species such as O(2)(-)* and *OH.

Repeated irradiation with suberythemal ultraviolet B reduces the number of epidermal Langerhans cells.

The influence of repeated low-dose ultraviolet B (UVB) radiation, to which we are exposed in daily life, has not been fully clarified, although the damage caused by exposure to high-dose UVB radiation has been well-studied in recent years. To investigate skin damage caused by repeated low-dose exposure, we evaluated the extent of injury to the Langerhans cells which are known to be involved in the cutaneous immune system. The backs of hairless mice were exposed to the following doses of UVB radiation: 100 mJ/cm(2) once, 50 mJ/cm(2) twice, 25 mJ/cm(2) four times or 10 mJ/cm(2) ten times. Skin specimens were taken for histochemical and electron microscopic examination 24 h after the final irradiation. Epidermis exposed to UVB radiation demonstrated a decrease in the number of Langerhans cells which showed less dendricity. The population of these cells in specimens exposed to repeated suberythemal doses was reduced to 40%, whereas exposure to a single high dose of UVB with the same energy resulted in a reduction of only 33%. These results indicate that repeated suberythemal doses injure Langerhans cells more than a single high-dose exposure. Furthermore, Birbeck granules in Langerhans cells of UVB-irradiated epidermis were reduced and tended to show shortening of their rod portion. The present study suggested that repeated challenge with suberythemal UVB radiation, to which we are all exposed in daily life, can cause substantial damage to Langerhans cells.

Production of singlet oxygen on irradiation of a photodynamic therapy agent, zinc-coproporphyrin III, with low host toxicity.

Zinc-coproporphyrin III (Zincphyrin) acts efficiently as a photodynamic therapy (PDT) agent in mice, while it shows no tumor cell-killing activity in vitro and has a high LD50 (low toxicity) in mice. It appears to have advantages over other porphyrins as a practical PDT reagent. In order to examine the action mechanism of Zincphyrin in PDT, we evaluated the photochemical characteristics of Zincphyrin by measurement of the near-infrared emission at 1268 nm, which provides direct evidence for formation of 1O2. Intense emission was observed in the presence of Zincphyrin, and was completely inhibited by NaN3, a 1O2 scavenger. Based on a quenching study, the rate constant of the reaction of 1O2 with NaN3 was determined to be 1.5-3.5 M(-1) s(-1), which is close to the reported value (3.8 x 10(8) M(-1) s(-1)). The intensity of the 1O2-specific emission was proportional to both the laser power and the concentration of Zincphyrin. The fluorescence quantum yield of Zincphyrin was 0.004 in phosphate buffer (100 mM, pH 7.4), which indicates that the excited state decays via other pathway(s) faster than through the fluorescence emission pathway. The lifetime of the triplet state of Zincphyrin (210 micros) was relatively long compared to that of other porphyrins, such as hematoporphyrin (Hp) (40 micros), coproporphyrin I (50 gs), or coproporphyrin III (36 gs). These results demonstrate the photodynamic generation of 1O2 by Zincphyrin.