Masako Naganuma

Topical trans-4-aminomethylcyclohexanecarboxylic acid prevents ultraviolet radiation-induced pigmentation

We have studied the effect of a plasmin inhibitor, trans-4-aminomethylcyclohexanecarboxylic acid (trans-AMCHA), on skin pigmentation induced by ultraviolet (UV) exposure in Weiser-Maples guinea pigs. When guinea pigs are exposed to UV radiation (840 mJ cm-2), skin pigmentation is clearly observed from seven days after exposure and continued to increase to 29 days. Post-exposure applications of 2 and 3% solutions of trans-AMCHA to the exposed regions prevent or inhibit the pigmentation process. When the skin is removed and stained by the Fontana-Masson method, melanin content in the basal layer of UV-exposed epidermis is significantly reduced in the regions to which 2 and 3% trans-AMCHA solutions have been applied, compared with the vehicle control. As plasmin is known to contribute to the release of arachidonic acid (AA) and the production of prostaglandins (PGs), we have examined the effects of trans-AMCHA on AA-induced pigmentation in guinea pig skin. Topical application of trans-AMCHA causes a dose-dependent decrease in AA-induced pigmentation. These results suggest that trans-AMCHA reduces melanocyte tyrosinase activity by suppressing the production of PGs, UV-induced melanogens, through the suppression of the UV-induced increase in epidermal plasmin activity.

EFFECTS OF CHRONIC EXPOSURE ULTRAVIOLET‐A INCLUDING 2% ULTRAVIOLET‐B ON FREE RADICAL REDUCTION SYSTEMS IN HAIRLESS MICE

Chronic ultraviolet (UV) irradiation is known to cause a variety of changes in the skin, including wrinkles, pigmented spots and carcinogenesis. To explore time dependent changes in several parameters with chronic UV irradiation, we examined the molecular changes in connective tissue, intracellular defence enzymes and free radical antioxidant substances in hairless mice skin caused by chronic exposure to UV‐A including 2% UV‐B. Connective tissue changes were estimated using hydroxyproline and isodesmosine assays as a measure of collagen and elastin concentrations, respectively.

Comparison of the melanogenesis in human black and light brown melanocytes

We examined how and to what extent the constitution of melanin and the expression, as well as the activity, of melanosomal proteins influence the production of melanin pigment by human black and light brown melanocytes, Mel (b) cells and Mel (l) cells, respectively. Melanin pigment in Mel (b) and Mel (l) cells consisted of a mixture of eumelanin and pheomelanin, and Mel (b) cells contained a larger amount. The signal intensity ratio of eumelanin to pheomelanin was similar in both cell types, though the two cell types differed in appearance. Tyrosinase activity and the amount of tyrosinase-related protein (TRP-1) of Mel (b) cells were higher than those of Mel (l) cells. Dopachrome tautomerase (DCT) activity and the amount of 6H5MICA were reduced in Mel (b) cells in comparison with Mel (l) cells. No significant difference in DHICA-converting activity or catechol-O-methyltransferase activity was found between Mel (b) and Mel (l) cells. There was no correlation between DHICA-converting activity and amount of TRP-1. These results suggest that the difference in the pigmentation of the two human melanocyte cell lines, Mel (b) and Mel (l), is derived from differences in the activity and expression of tyrosinase, TRP-1 and DCT, which affect the content and constitution of melanin polymers.

Algorithm for in vitro Sun Protection Factor Based on Transmission Spectrum Measurement with Concomitant Evaluation of Photostability

In thein vitro evaluation of Sun Protection Factor (SPF), the photostability of the ultraviolet (UV) filters can have a major impact, especially for high‐SPF formulations, but is generally not taken into consideration. In this study, we present a UV transmission spectrum measurement system utilizing a high‐sensitivity UV photomultiplier tube with concomitant evaluation of photostability. We have developed an algorithm to estimate SPF in vitro by converting the amount of UV light transmission through the sunscreen layer into cumulative relative erythema effectiveness to obtain one minimal erythema dose. Thus, the algorithm uses the same endpoint as in vivo SPF methods, but with a photomultiplier tube as the detector instead of skin. The values obtained showed an excellent correlation with in vivo SPF values, even for high‐SPF sunscreens exceeding SPF 50. This method should be suitable as an in vitro SPF testing method for regulatory purposes.

The Proliferation and Differentiation of Neonatal Epidermal Melanocytes in F1 Hairless Mice of HR‐1 × HR/De in Serum‐Free Culture

To investigate the characteristics of the proliferation and differentiation of epidermal melanocytes in F1 hairless mice of HR‐1 × HR/De parents in vitro, cell suspensions of the neonatal epidermis were cultured in a serum‐free medium supplemented with dibutyryl adenosine 3‘,5‘‐cyclic monophosphate (DBcAMP) and/or basic fibroblast growth factor (bFGF). The differentiation of melanocytes was induced by treatment with DBcAMP. In contrast, the sustained proliferation of melanoblasts was induced by combined treatment with DBcAMP and bFGF. The melanoblasts could be subcultured in serum‐free medium supplemented with the two factors in the presence of keratinocytes, but not in their absence. This is the first report of successful culture of melanoblasts and melanocytes from hairless mice; it is expected to be useful in understanding the mechanism of the development of pigmented spots in the epidermis of (HR‐1 × HR/De) F1 mice, which are reported to be induced by repeated exposure to ultraviolet light B.

Phospholipases Induce Melanogenesis in Organ‐Cultured Skin

Guinea pig skin becomes more pigmented following exposure to UV rays. This melanization was accompanied by enhanced intensity of tyrosinase‐staining and increased number of tyrosinase‐positive melanocytes (MELty+), with resultant enhancement of melanin synthesis. To clarify the regulatory mechanism for melanization following UV irradiation, organ‐cultured guinea pig skins have been used to examine their melanogenic responses to exogenous stimulation. This organ culture system responded well to UV irradiation, by increasing melanogenic activity. Also, in this system, phospholipases (PL), arachidonic acid, interleukin‐1α and melanocytestimulating hormone, but not endothelin‐1 or phosphatidylinositol‐specific PLC (PI‐PLC), stimulated melanogenesis to various extents as indicated by the number of MELty+ and morphological changes. Among them, the PLA2 and PLD were found to have a potent stimulatory property for melanocytes. They might affect melanocytes directly or indirectly through an effect on keratinocytes. These results suggest that PLA2 and PLD play a key role in epidermal hyperpigmentation after UV irradiation or inflammation.

PHOTOTOXIC REACTION TO XANTHENE DYES INDUCED BY VISIBLE LIGHT

Many dyes, for instance methylene blue, rose bengal, and eosin, are known as photosensitizers, and in the presence of molecular oxygen they induce cell lethality and skin photosensitivity (1–4). Several dyes are used in cosmetic products, particularly in lipsticks. Human lip skin is therefore exposed to potential danger from dye‐sensitized phototoxic reactions. Using an in vivo system of mammalian skin, such as the abdominal skin of rabbits, we established screening tests for the phototoxic potential of synthetic dyes in two ways: (a) intracutaneous injection; (b) topical application with and without damaging the barrier property of the stratum corneum.

MELANOCYTE-STIMULATING PROPERTIES OF SECRETORY PHOSPHOLIPASE A2

Abstract— Phospholipase A2 (PLA2) catalyzes the release of free fatty acids from membrane phospholipids, and its products derived from these fatty acids, such as prostaglandins and leukotrienes, significantly up‐regulate the key mela‐nogenic enzyme, tyrosinase, in melanocytes. This has led to suggestions that PLA2 itself triggers melanin synthesis in melanogenesis following UV irradiation or inflammation.