Ebselen is a new skin depigmenting agent that inhibits melanin biosynthesis and melanosomal transfer

We assessed the ability of ebselen, a glutathione peroxidase mimic, to reduce pigmentation in various models. In murine B16 melanocytes, 25 μm ebselen inhibited melanogenesis and induced a depolymerisation of actin filaments. In co-cultures of B16 melanocytes with BDVII keratinocytes, a pretreatment of melanocytes with ebselen resulted in a strong inhibition of melanosome transfer to keratinocytes, as shown under optical and electron microscopy. In reconstructed epidermis, topical 0.5% ebselen led to a twofold decrease of melanin without affecting the density of active melanocytes. A similar result was obtained with topical 0.5% ebselen in black guinea pig ears. Ebselen induced a decrease of epidermal melanin parallel to a localisation of melanin and melanosomes in the basal layer. Ebselen appears as a new depigmenting compound that inhibits melanin synthesis and melanosome transfer to keratinocytes.     

Infrared A radiation promotes survival of human melanocytes carrying ultraviolet radiation‐induced DNA damage

The link between solar radiation and melanoma is still elusive. Although infrared radiation (IR) accounts for over 50% of terrestrial solar energy, its influence on human skin is not well explored. There is increasing evidence that IR influences the expression patterns of several molecules independently of heat. A previous in vivo study revealed that pretreatment with IR might promote the development of UVR‐induced non‐epithelial skin cancer and possibly of melanoma in mice. To expand on this, the aim of the present study was to evaluate the impact of IR on UVR‐induced apoptosis and DNA repair in normal human epidermal melanocytes. The balance between these two effects is a key factor of malignant transformation. Human melanocytes were exposed to physiologic doses of IR and UVR. Compared to cells irradiated with UVR only, simultaneous exposure to IR significantly reduced the apoptotic rate. However, IR did not influence the repair of UVR‐induced DNA damage. IR partly reversed the pro‐apoptotic effects of UVR via modification of the expression and activity of proteins mainly of the extrinsic apoptotic pathway. In conclusion, IR enhances the survival of melanocytes carrying UVR‐induced DNA damage and thereby might contribute to melanomagenesis.     

Efficacy of quantifying melanosome transfer with flow cytometry in a human melanocyte–HaCaT keratinocyte co‐culture system in vitro

Please cite this paper as: Efficacy quantifying melanosome transfer with flow cytometry in a human melanocyte–HaCaT keratinocyte co‐culture system in vitro. Experimental Dermatology 2010; 19 : e282–e285. Abstract: In this study, we describe a simple, specific, reproducible and quantitative assay system to assess melanosome transfer. We first established a co‐culture model of normal human epidermal melanocytes and HaCaT keratinocytes. The cells were co‐cultured for 72 h in a serum‐free keratinocyte growth media and double labelled with Fluorescein isothiocyanate (FITC)‐conjugated antibody against the melanosome‐specific protein gp100, and with Phycoerythrin (PE)‐conjugated antibody against the keratinocyte‐specific marker cytokeratin. Then, the cells were examined using co‐focal microscope and flow cytometry. The increased melanosome transfer from melanocytes to HaCaT keratinocytes was observed in a time‐dependent manner. To verify the accessibility of this method, two known melanosome transfer inhibitors and two known melanosome transfer stimulators were applied. Consistent with previous investigation, soybean trypsin inhibitor (STI), niacinamide inhibited melanosome transfer, α‐melanocyte stimulating hormone (α‐MSH) and keratinocyte growth factor (KGF) increased melanosome transfer, respectively, in a dose‐dependent manner. The model used in this study could thus represent a rapid and reliable tool to identify modulators of human melanosome transfer.     

Alterations in the epidermal-dermal melanin axis and factor XIIIa melanophages in senile lentigo and ageing skin

Background Senile lentigo (SL) is a pigmentation disorder that occurs predominantly on the dorsa of the hands, the forearms and the face; its incidence increases with age. Histological hallmarks of SL lesions are hyperpigmentation of the epidermis and elongation of the epidermal rete ridges. Various factors such as α‐melanocyte‐stimulating hormone, endothelin‐1 or stem cell factor are involved in the onset and maintenance of the increased pigmentation. Alterations of the dermal compartment have not yet been analysed in detail in SL. Objectives To study the occurrence and distribution of melanin in the dermis from SL and aged skin, biopsies from 12 subjects were morphologically analysed by light and electron microscopy in comparison with unaffected skin. Methods Punch biopsies of SL and adjacent skin from 12 male or female volunteers aged 52–81 years were prepared for light and electron microscopy and samples were analysed by morphological, morphometric, histochemical and immunohistochemical methods. Results The epidermis from SL revealed morphological features such as hyperpigmentation of basal keratinocytes and the formation of elongated rete ridges. S100+ melanocytes in the stratum basale were not markedly increased, indicating that the hyperpigmentation is predominantly due to changes in melanin synthesis, distribution or turnover. Quantification of epidermal cells expressing the proliferation marker Ki67 did not show an increase of this parameter in SL, indicating that at least in the established lesion cell proliferation is not enhanced. We further focused on the dermal compartment and observed granulated cells which were more abundant in SL. Electron microscopic and histochemical analysis revealed that the granulation of these cells is based on melanosomes, mostly present in large melanosomal complexes. Immunohistochemistry using antibodies to CD68 and factor XIIIa (FXIIIa) showed these melanophages to be predominantly FXIIIa+ dermal dendrocytes, which were about six times more abundant than CD68+ macrophages. Conclusions In SL an increased number of melanophages was found compared with unaffected skin from the same subject. These melanophages were identified as FXIIIa+ dermal dendrocytes. Possible functional consequences of the massive melanin uptake by dermal dendrocytes are discussed.     

Placental extracts regulate melanin synthesis in normal human melanocytes with alterations of mitochondrial respiration

Placental extracts have been widely used as skin lightening agents in the Japanese cosmetic market. Here, we show that placental extracts contain factors that can decrease or increase melanin synthesis by normal human melanocytes in vitro in possible association with mitochondrial respiration. When normal human melanocytes were treated with a whole porcine placental extract, melanin synthesis was decreased. In contrast, a porcine placental extract in which exudates and insoluble materials including lipids had been removed increased melanin synthesis. In addition, the amount of tyrosinase, the enzyme critical for melanin synthesis, changed in accordance with the alteration of melanin synthesis. Interestingly, the amount of manganese‐dependent superoxide dismutase (MnSOD), a mitochondrial‐resident antioxidant enzyme, was increased when melanin synthesis was decreased by the whole placental extract. Mitochondrial respiration and glycolysis also changed following treatment with the placental extracts. These results suggest that placental extracts contain factors that can increase or decrease melanin synthesis by normal human melanocytes and that mitochondrial function may be associated with the placental extract‐induced regulation of melanogenesis.     

The effects of Sophora angustifolia and other natural plant extracts on melanogenesis and melanin transfer in human skin cells

Skin pigmentation is a multistep process of melanin synthesis by melanocytes, its transfer to recipient keratinocytes and its degradation. As dyspigmentation is a prominent marker of skin ageing, novel effective agents that modulate pigmentation safely are being sought for both clinical and cosmetic use. Here, a number of plant extracts were examined for their effect on melanogenesis (by melanin assay and Western blotting) and melanin transfer (by confocal immunomicroscopy of gp100‐positive melanin granules in cocultures and by SEM analysis of filopodia), in human melanocytes and in cocultures with phototype‐matched normal adult epidermal keratinocytes. Mulberry, Kiwi and Sophora extracts were assessed against isobutylmethylxanthine, hydroquinone, vitamin C and niacinamide. Compared with unstimulated control, all extracts significantly reduced melanogenesis in human melanoma cells and normal adult epidermal melanocytes. These extracts also reduced melanin transfer and reduced filopodia expression on melanocytes, similar to hydroquinone and niacinamide, indicating their effectiveness as multimode pigmentation actives.     

Blue light inhibits melanin synthesis in B16 melanoma 4A5 cells and skin pigmentation induced by ultraviolet B in guinea-pigs

BACKGROUND: Little has been known about the effects of visible light in mammalian cells. We recently found that blue light not only suppressed the growth of B16 melanoma cells in a time-dependent manner but also inhibited metastasis of the B16 melanoma cells to the lung. These findings suggest that exposure to blue light modifies the functions of B16 melanoma cells. The present study investigated the effects of blue light on B16 melanoma 4A5 cells and Weiser-Maple guinea-pigs to confirm the biological effect of blue light on melanin formation. METHODS: The effect of red, green, and blue light on melanin synthesis in B16 melanoma 4A5 cells was measured. The back skin of brown Weiser-Maple guinea-pigs was exposed to ultraviolet B (UVB; 588 mJ/cm(2) (0.7 mW/cm(2)x 14 min) three times a week for 2 weeks to induce melanin deposition. Thirty minutes after each UVB exposure, blue light was applied for 30 min. Pigmentation of the exposed areas of skin was checked once a week, and photographs of the skin were taken by digital camera. Observation was continued for 18 days after the final UVB exposure. RESULTS: Melanin synthesis in B16 melanoma 4A5 cells was selectively suppressed by blue light, but blue light did not induce decolorization of previously produced melanin. In the back skin of brown guinea-pigs, the brightness of the sites exposed to UVB began to decrease on the fifth day of the experiment, decreasing further from the 12th day to the 18th day after UVB exposure. The brightness of the sites exposed to UVB and blue light decreased in a manner similar during the UVB exposure, but remained relatively unchanged from the 12th day to the 30th day. CONCLUSIONS: These results suggest that blue light suppresses melanin formation following repeated UVB exposure. Further investigation with various light such as blue light may lead to a new approach to the care of ultraviolet-affected skin such as hyperpigmentation.     

Hyperbaric oxygen improves ultraviolet B irradiation‐induced melanin pigmentation and diminishes senile spot size

Background: The effects of exposure to hyperbaric oxygen on ultraviolet B (UVB) irradiation‐induced melanin pigmentations of skins and on senile spot sizes of faces were investigated. Methods: In the first experiment, male subjects were irradiated with UVB on their upper arms for inducing erythema and the subsequent melanin pigmentation. They were exposed to a hyperbaric environment at 1.25 atmospheres absolute (ATA) with 32% oxygen for 1 h/day, three times per week. In the second experiment, female subjects were exposed to a hyperbaric environment at 1.25 ATA with 32% oxygen for 1 h/day, two times per week. Results: In the first experiment, melanin pigmentations lightened after 4 weeks of exposure to hyperbaric oxygen. In the second experiment, senile spot sizes became small after 12 weeks of exposure to hyperbaric oxygen. Conclusion: We concluded that exposure to hyperbaric oxygen used in this study accelerates both the fading in melanin pigmentation and the decrease in senile spot size.     

Co-culture of human melanocytes and keratinocytes in a skin equivalent model: effect of ultraviolet radiation

Melanocytes grown in pure monolayer culture lack the three-dimensional organization and many of the cellular interactions that exist in vivo. This can be partially overcome by growing melanocytes together with other epidermal cells in skin equivalent models. In this study skin equivalents were prepared by seeding mixtures of cultured human keratinocytes and melanocytes in various ratios onto de-epidermized dermis. They were cultured in DMEM/Ham's F12 (31) for 3 days and then lifted to the air-liquid interface and maintained for 11 days. Histological examination revealed a structure that closely resembled human interfollicular epidermis. Melanocytes, identified by their dendritic appearance, positive dopa reaction and positive staining with a melanocyte-specific antibody (MEL5), were located in the basal layer. Melanin was seen both in melanocytes and in neighbouring keratinocytes. Whilst the skin equivalent became more pigmented following UV irradiation (total UVB 4760 J/m² over 3 days), the quantity and distribution of melanin at the light microscopic level appeared to be unchanged. However, the number and dendricity of melanocytes increased, as did their staining with dopa and MEL5. These results indicate that melanocytes are functional and capable of responding to UV irradiation.     

Non‐invasive diffuse reflectance measurements of cutaneous melanin content can predict human sensitivity to ultraviolet radiation

The diversity of human skin phenotypes and the ubiquitous exposure to ultraviolet radiation (UVR) underscore the need for a non‐invasive tool to predict an individual's UVR sensitivity. We analysed correlations between UVR sensitivity, melanin content, diffuse reflectance spectroscopy (DR) and UVR‐induced DNA damage in the skin of subjects from three racial/ethnic groups: Asian, Black or African American and White. UVR sensitivity was determined by evaluating each subject's response to one minimal erythemal dose (MED) of UVR one day after the exposure. Melanin content was measured using DR and by densitometric analysis of Fontana‐Masson staining (FM) in skin biopsies taken from unexposed areas. An individual's UVR sensitivity based on MED was highly correlated with melanin content measured by DR and by FM. Therefore, a predictive model for the non‐invasive determination of UVR sensitivity using DR was developed. The MED precision was further improved when we took race/ethnicity into consideration. The use of DR serves as a tool for predicting UVR sensitivity in humans that should be invaluable for determining appropriate UVR doses for therapeutic, diagnostic and/or cosmetic devices.     

Photoprotection of bacterial-derived melanin against ultraviolet A–induced cell death and its potential application as an active sunscreen

Background  The increase in the incidence of non-melanoma skin tumours, photoaging, and immunosuppression demand for more effective sunscreen on ultraviolet A (UVA) irradiation.
Objectives  The aim of the study is to evaluate the photoprotective effects of a bacterial-derived melanin against UVA-induced damages in vitro and in vivo .
Methods  Human fibroblasts were used to assess the role of the bacterial-derived melanin on cell viability against UVA. 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay and nuclear morphology were employed to evaluate the photoprotection at the cellular level. Fluorometric assays were performed to detect the formation of reactive oxygen species (ROS) in the cells. Evaluations of the bacterial-derived melanin as a sunscreen were measured by transmission test and persistent pigment darkening on human skin.
Results  Bacterial-derived melanin efficiently scavenged ROS in the fibroblasts after UVA irradiation. The cell viability of xeroderma pigmentosum (XP) fibroblast treated with varied doses of melanin increased dramatically in comparison with untreated control and the treated XP fibroblasts became more resistant to UVA-induced apoptosis than normal fibroblasts. Although the relative transmission didn't change too much with different concentration of bacterial-derived melanin, this melanin could keep UVA-irradiated skin from pigment darkening and act as an active sunscreen on skin.
Conclusions  The bacterial-derived melanin provided significant protection to fibroblast cell and human skin against the UVA radiation. It has the potential to be developed as an active sunscreen for the patients with photosensitivity skin to sun exposure.     

Non‐cultured melanocyte‐keratinocyte transplantation for the treatment of vitiligo: a clinical trial in an Iranian population

Background Non‐cultured cellular grafting as a surgical procedure is indicated to treat stable vitiligo, refractory to medical treatment, and is gaining wider acceptance among dermatologists. Objective The aim of this study was to assess the efficacy of non‐cultured melanocyte‐keratinocyte transplantation (MKT) for the treatment of generalized vitiligo in Iranian patients. Methods In this clinical trial, a total of 14 vitiligo patches in eight patients were treated; eight patches with non‐cultured MKT and six patches dermabraded alone without application of keratinocyte‐melanocyte suspension. Repigmentation was compared at about 4 months post‐transplantation. Results Of the eight lesions treated with non‐cultured MKT, four lesions showed 96–100% repigmentation, one lesion 65–95% and three lesions 0–25%. Of the patients who showed excellent results, only one showed a post‐inflammatory hyperpigmentation in recipient and control areas. Of the six control lesions, five showed failed repigmentation and one showed post‐inflammatory hyperpigmentation. Conclusion Non‐cultured MKT is an effective method to treat stable vitiligo. Studies on larger series of vitiligo patients are required to confirm its efficacy.     

The photoadaptive response to ultraviolet exposure in human skin using ultraviolet spectrophotometry

BACKGROUND: Both pigmentation and non-pigmentary processes contribute to the development of photoadaptation yet the exact contribution of either in the resting state and in response to ultraviolet (UV) radiation is unclear. The purpose of this study was to estimate independently these changes occurring in the epidermis following repeated exposure to UV in two groups with differing degrees of constitutive pigmentation. METHODS: We describe a mathematical model for explaining the spectral absorbance of excised human epidermis based on the absorbance of constituent chromophores. The model was applied to spectral absorbance data measured on samples of epidermis excised from pre-irradiated skin and from skin obtained following UV irradiation on 3 successive days. RESULTS: We found that in Asian skin there was only a mild photoadaptive response, principally by a small increase in pigmentation. On the other hand, the significant adaptive response in Caucasian skin was through hyperplasia of the epidermis, with tanning contributing only to a much smaller degree. CONCLUSION: This study has enabled us to study independently the pigmentary and non-pigmentary pathways and has shown that in those people with a lower degree of constitutive pigment, the primary mechanism of photoadaptation is via the non-pigmentary route.     

Differential expression of inflammatory cytokines and chemokines in lipopolysaccharide‐stimulated melanocytes from lightly and darkly pigmented skin

Increasing evidence suggests that human epidermal melanocytes play an important role in the skin immune system; however, a role of their pigmentation in immune and inflammatory responses is poorly examined. In the study, the expression of Toll‐like receptor 4 (TLR4) and inflammatory cytokines and chemokines by cultured normal melanocytes derived from lightly and darkly pigmented skin was investigated after cell stimulation with lipopolysaccharide (LPS). The basal TLR4 mRNA level in heavily pigmented cells was higher as compared to their lightly pigmented counterparts. Melanocyte exposure to LPS upregulated the expression of TLR4 mRNA and enhanced the DNA‐binding activity of NF‐κB p50 and p65. We found substantial differences in the LPS‐stimulated expression of numerous genes encoding inflammatory cytokines and chemokines between the cells with various melanin contents. In lightly pigmented melanocytes, the most significantly upregulated genes were nicotinamide phosphoribosyltransferase (NAMPT/visfatin), the chemokines CCL2 and CCL20, and IL6, while the genes for CXCL12, IL‐16 and the chemokine receptor CCR4 were the most significantly upregulated in heavily pigmented cells. Moreover, the lightly pigmented melanocytes secreted much more NAMPT, CCL2 and IL‐6. The results of our study suggest modulatory effect of melanogenesis on the immune properties of normal epidermal melanocytes.     

Galanin contributes to ultraviolet irradiation‐induced inflammation in human skin

Exposure of the skin to ultraviolet (UV) irradiation causes various consequences such as inflammation and photoageing. Galanin is an active neuropeptide expressed widely in the central nervous system and peripheral tissues including the skin. Galanin promotes or inhibits inflammation in a context‐dependent manner, but its role in UV irradiation‐induced responses in human skin was still unknown. UV irradiation induced a substantial expression of galanin in primary epidermal keratinocytes in vitro and in human epidermis in vivo. Galanin knock‐down by siRNA transfection markedly inhibited UV irradiation‐induced expression of matrix metalloproteinase (MMP)‐1, interleukin (IL)‐1β, IL‐6 and cyclooxygenase (COX)‐2. Moreover, siRNA‐mediated knock‐down of GAL₂, a principal galanin receptor in the skin, led to a considerable decrease in these mediators in keratinocytes. Collectively, our findings suggest that galanin is an important messenger between the neuroendocrine system and UV irradiation‐damaged skin.     

Analysis of ionizing radiation‐induced DNA damage and repair in three‐dimensional human skin model system

Please cite this paper as: Analysis of ionizing radiation‐induced DNA damage and repair in three‐dimensional human skin model system. Experimental Dermatology 2010; 19 : e16–e22. Abstract: Knowledge of cellular responses in tissue microenvironment is crucial for the accurate prediction of human health risks following chronic or acute exposure to ionizing radiation (IR). With this objective, we investigated the radio responses for the first time in three‐dimensional (3D) artificial human skin tissue microenvironment after γ‐rays radiation. IR‐induced DNA damage/repair response was assessed by immunological analysis of well‐known DNA double strand break (DSB) repair proteins, i.e. 53BP1 and phosphorylated ataxia telangiectasia mutated^ser1981 (ATM^ser1981). Efficient 53BP1 and phosphorylated ATM foci formation was observed in human EpiDerm tissue constructs after low and high doses of γ‐rays. Interestingly, EpiDerm tissue constructs displayed less 53BP1 and ATM foci number at all radiation doses (0.1, 1, 2.5 and 5 Gy) than that observed for 2D human fibroblasts. DSB repair efficiency judged by the disappearance of 53BP1 foci declined with increasing doses of γ‐rays and tissue constructs irradiated with 2.5 and 5 Gy of γ‐rays displayed 53BP1 foci persisting up to 72 h of analysis. Pretreatment of EpiDerm tissue constructs with LY294002, [an inhibitor of phosphatidylinositol‐3 kinase and PI‐3 kinase like kinases (PIKK)] completely abolished IR‐induced 53BP1 foci formation and increased the apoptotic death. This observation indicates the importance of PIKK signalling pathway for efficient radiation responses in intact tissue constructs. In summary, we have successfully demonstrated the feasibility of monitoring the DNA damage response in human skin tissue microenvironment. In this system, 53BP1 can be used as a useful marker for monitoring the DSB repair efficiency.     

How hormones may modulate human skin pigmentation in melasma: An in vitro perspective

Melasma is a common acquired hyperpigmentary disorder occurring primarily in photo‐exposed areas and mainly affecting women of childbearing age. To decipher the role of sex hormones in melasma, this viewpoint reviews the effects of sex hormones on cutaneous cells cultured in monolayers, in coculture, in 3D models and explants in the presence or the absence of UV. The data show that sex steroid hormones, especially oestrogen, can modulate in vitro pigmentation by stimulating melanocytes and keratinocyte pro‐pigmentary factors, but not via fibroblast or mast cell activation. In vitro data suggest that oestrogen acts on endothelial cell count, which may in turn increase endothelin‐1 concentrations. However, data on explants revealed that sex steroid even at doses observed during pregnancy cannot induce melanogenesis alone nor melanosome transfer but that it acts in synergy with UVB. In conclusion, we hypothesize that in predisposed persons, sex steroid hormones initiate hyperpigmentation in melasma by amplifying the effects of UV on melanogenesis via direct effects on melanocytes or indirect effects via keratinocytes and on the transfer of melanosomes. They also help to sustain hyperpigmentation by increasing the number of blood vessels and, in turn, the level of endothelin‐1.