Melanin accumulation in acanthotic seborrheic keratosis: Reduced proliferation and early differentiation of keratinocytes and increased number of melanocytes.

Seborrheic keratosis (SK) is a common benign tumour, often associated with hyperpigmentation. To investigate the mechanism of melanin accumulation in SK, we have conducted comprehensive gene expression and histological analyses. We obtained five pairs of skin samples, including non-lesional and SK samples, from the backs of three male Japanese participants aged 40-59 years. To examine melanocytes and keratinocytes in SK, three pairs of skin samples were separated by laser capture microdissection into the basal layer and the other layer in the epidermis. We performed a comprehensive gene expression analysis to identify differentially expressed genes between non-lesional and SK skin, followed by gene ontology and pathway analysis. We found abnormal morphogenesis and cell proliferation in the basal layer, along with increased immune response and impaired cell differentiation and metabolism in the other layer of SK. We focused on cell proliferation and differentiation, as these are directly associated with melanin accumulation. Immunohistochemical analyses of Ki67, keratin 10, and keratin 14 demonstrated the decreases in the proliferation and early differentiation of the epidermis. Contrarily, no significant changes were observed in terminal differentiation markers, filaggrin and loricrin. Although the number of melanocytes was higher in SK than in non-lesional skin, melanogenic activity showed no difference. These results indicated that melanin accumulation in SK is caused by delayed melanin excretion due to reduced turnover around the basal and spinous layers of the epidermis and melanin production due to an increased number of melanocytes. Our findings provide new insights for therapeutic approaches in SK.

Efficacy of a fine fiber film applied with a water-based lotion to improve dry skin.

BACKGROUND: Dry skin can trigger eczema that affects >10% of the US population. Dressing films have been developed to improve diseased skin, but there is limited knowledge about their effects, especially for dry skin-related symptoms. We developed an electrospinning method that creates a coating film, called a fine fiber (FF) film, characterized by the production of a transparent, thin, flexible, and adherent membrane on the skin surface. OBJECTIVE: The aim of this pilot study was to examine the effects of the FF film on dry skin. METHODS: Three treatments (lotion only, lotion with the FF film, and lotion with an alternative film) were designed to treat subjects with rough skin on their lower legs. Twenty-four females were enrolled and used either a water-based lotion U or a petrolatum-based lotion P and the FF film for 2 weeks followed by a regression phase for 1 week. Skin hydration and roughness scores were assessed as were the subjects' perceptions of the effects. RESULTS: When the FF film was applied with lotion U, skin hydration was significantly improved even after 1 week, accompanied by a significant improvement of skin roughness and an increase in skin hydration by the end of the regression phase. An evaluation of moisture permeability suggested that the FF film, especially with lotion U, performed as a semipermeable membrane with optimal moisture healing effects on dry skin. CONCLUSION: The FF film together with a water-based lotion is a promising treatment to quickly improve dry skin conditions.

Exploitation of long-lasting ultraweak photon emission to estimate skin photodamage after ultraviolet exposure.

BACKGROUND: Establishing a noninvasive method to estimate skin damage immediately after ultraviolet (UV) exposure is required to minimize the anticipated severe symptoms triggered by early phase UV-induced reactions in the skin. To develop a suitable method, we focused on ultraweak photon emission (UPE) immediately after UV exposure to characterize the relationship of UPE to skin photodamage caused by the UV exposure. MATERIALS AND METHODS: Analysis of the correlation between UV-induced UPE and erythema formation characterized by skin redness was conducted in a clinical study. To clarify the source of UPE, time-dependent lipid oxidation was analyzed in human epidermal keratinocytes in vitro using a fluorescence indicator as well as the lipid hydroperoxide (LPO) assay. RESULTS: The average amount of UV-induced long-lasting UPE per second, especially from 1 to 3 minutes compared to other time periods after the UV radiation, increased in a dose-dependent manner and was highly correlated with the intensity of cutaneous redness 24 hours after UV exposure. In addition, cellular examinations elucidated that both the long-lasting UPE signals and the increased amounts of LPO 2 minutes after UV radiation were significantly suppressed by Trolox (a vitamin E derivative), which has been shown to inhibit UV-induced erythema formation in human skin. CONCLUSION: Long-lasting UPE generated between 1 and 3 minutes immediately after UV exposure, which is associated with LPO production, is a valuable indicator to estimate and/or avoid severe cutaneous photodamage.

Evaluation of subclinical chronic sun damage in the skin via the detection of long-lasting ultraweak photon emission.

BACKGROUND: It is well known that solar radiation accelerates skin photoaging. To evaluate subclinical photodamage in the skin especially from the early phase of ultraviolet (UV)-induced damage, we have focused on ultraweak photon emission (UPE), also called biophotons. Our previous study reported that the amount of long-lasting UPE induced by UV, predominantly from lipid peroxidation, is a valuable indicator to assess cutaneous photodamage even at a suberythemal dose, although it was only applied to evaluate acute UV damage. The aim of this study was to further investigate whether long-lasting UPE could also be a useful marker to assess subclinical chronic sun damage in the course of skin photoaging. MATERIALS AND METHODS: Forty-three Japanese females in their 20s were recruited and were divided into two groups according to their history of sun exposure based on a questionnaire (high- and low-sun-exposure groups). Several skin properties on the cheek and outer forearm were measured in addition to UV-induced UPE. RESULTS: Among the skin properties measured, water content, average skin roughness, and the lateral packing of lipids in the stratum corneum were significantly deteriorated in the high-sun-exposure group as were changes in some skin photoaging scores such as pigmented spots and wrinkles. In addition, those skin properties were correlated with the UPE signals, suggesting the possible impact of oxidative stress on chronic skin damage. CONCLUSION: Subtle oxidative stress detected by long-lasting UPE may contribute to subclinical cutaneous damage at the beginning phase of chronic sun exposure, which potentially enhances skin photoaging over a lifetime.

Generation of hydroxyl radicals and singlet oxygen during oxidation of rhododendrol and rhododendrol-catechol.

The generation of hydroxyl radicals and singlet oxygen during the oxidation of 4-(4-hydroxyphenyl)-2-butanol (rhododendrol) and 4-(3,4-dihydroxyphenyl)-2-butanol (rhododendrol-catechol) with mushroom tyrosinase in a phosphate buffer (pH 7.4) was examined as the model for the reactive oxygen species generation via the two rhododendrol compounds in melanocytes. The reaction was performed in the presence of 5,5-dimethyl-1-pyrroline-N-oxide (DMPO) spin trap reagents for hydroxyl radical or 2,2,6,6-tetramethyl-4-piperidone (4-oxo-TEMP), an acceptor of singlet oxygen, and their electron spin resonances were measured. An increase in the electron spin resonances signal attributable to the adduct of DMPO reacting with the hydroxyl radical and that of 4-oxo-TEMP reacting with singlet oxygen was observed during the tyrosinase-catalyzed oxidation of rhododendrol and rhododendrol-catechol, indicating the generation of hydroxyl radical and singlet oxygen. Moreover, hydroxyl radical generation was also observed in the autoxidation of rhododendrol-catechol. We show that generation of intermediates during tyrosinase-catalyzed oxidation of rhododendrol enhances oxidative stress in melanocytes.

Substantial evidence for the rhododendrol-induced generation of hydroxyl radicals that causes melanocyte cytotoxicity and induces chemical leukoderma.

BACKGROUND: Rhododendrol (4-(4-hydroxyphenyl)-2-butanol) has been used as a lightening/whitening cosmetic but was recently reported to induce leukoderma. Although rhododendrol has been shown to be transformed by tyrosinase to hydroxyl-rhododendrol, which is cytotoxic to melanocytes, its detailed mechanism of action including the involvement of reactive oxygen species is not clearly understood. OBJECTIVE: To confirm the relationship of hydroxyl radical generation to melanocyte cytotoxicity induced by rhododendrol, this study was performed. METHODS: An electron spin resonance method with a highly sensitive detection system was utilized to monitor hydroxyl radicals generated from two distinct normal human epidermal melanocyte lines with different levels of tyrosinase activity after the addition of various amounts of rhododendrol. Cytotoxicity of rhododendrol was analyzed by AlamarBlue assay under the same condition. RESULTS: Hydroxyl radicals were generated depending on the amounts of rhododendrol and/or tyrosinase. After the correlation between hydroxyl radical generation with melanocyte viability was confirmed, an inhibitor of oxidative stress, N-acetyl cysteine, was shown to dramatically diminish rhododendrol-induced generation of hydroxyl radicals and melanocyte cytotoxicity by increasing glutathione levels. In contrast, buthionine sulfoximine, which depletes glutathione, augmented both of those parameters. CONCLUSION: Suppressing oxidative stress would prevent and/or mitigate some phenol derivative-induced leukoderma by avoiding hydroxyl radical-initiated melanocyte cytotoxicity.

Tunable design strategy for fluorescence probes based on 4-substituted BODIPY chromophore: improvement of highly sensitive fluorescence probe for nitric oxide.

4,4-Difluoro-4-bora-3a,4a-diaza-s-indacene (BODIPY) is a well-known fluorophore, with a high molar extinction coefficient and high fluorescence quantum efficiency (Phi(fl)). Furthermore, its structure can be modified to change its excitation and emission wavelengths. However, little work has been done on the structural modification of fluorines at the B-4 position with other functional groups. We synthesized 4-methoxy-substituted BODIPY derivatives in satisfactory yields, and found that they exhibited improved solubility in aqueous solution. Moreover, their oxidation and reduction potentials were greatly decreased without any change in their absorbance and fluorescence properties. These features of 4-substituted BODIPYs may be useful for developing novel fluorescence probes based on the intramolecular photoinduced electron transfer (PeT) mechanism, because it is possible to optimize the PeT process precisely by modulating the electrochemical properties of the fluorophore. The value of this approach is exemplified by its application to the development of a highly sensitive and pH-independent fluorescence probe for nitric oxide.

Highly sensitive fluorescence probes for nitric oxide based on boron dipyrromethene chromophore-rational design of potentially useful bioimaging fluorescence probe.

Boron dipyrromethene (BODIPY) is known to have a high quantum yield (phi) of fluorescence in aqueous solution but has not been utilized much for biological applications, compared to fluorescein. We developed 8-(3,4-diaminophenyl)-2,6-bis(2-carboxyethyl)-4,4-difluoro-1,3,5,7-tetramethyl-4-bora-3a,4a-diaza-s-indacene (DAMBO-P(H)), based on the BODIPY chromophore, as a highly sensitive fluorescence probe for nitric oxide (NO). DAMBO-P(H) had a low phi value of 0.002, whereas its triazole derivative (DAMBO-P(H)-T), the product of the reaction of DAMBO-P(H) with NO, fluoresced strongly (phi = 0.74). The change of the fluorescence intensity was found to be controlled by an intramolecular photoinduced electron transfer (PeT) mechanism. The strategy for development of DAMBO-P(H) was as follows: (1) in order to design a highly sensitive probe of NO, the reactivity of o-phenylenediamine derivatives as NO-reactive moieties was examined using 4,5-diaminofluorescein (DAF-2, a widely used NO fluorescence probe), (2) in order to avoid pH-dependency of the fluorescence intensity, the PeT process was controlled by modulating the spectroscopic and electrochemical properties of BODIPY chromophores according to the Rehm-Weller equation based on measurement of excitation energies of chromophores, ground-state reduction potentials of PeT acceptors (BODIPYs), and calculation of the HOMO energy level of the PeT donor (o-phenylenediamine moiety) at the B3LYP/6-31G level, (3) in order to avoid quenching of fluorescence by stacking of the probes and to obtain probes suitable for biological applications, hydrophilic functional groups were introduced. This strategy should be applicable for the rational design of other novel and potentially useful bioimaging fluorescence probes.