Assessment of ultraviolet‐radiation‐induced DNA damage within melanocytes in skin of different constitutive pigmentation

Background Melanoma incidence and pigmentary disorders are known to be related to the degree of skin pigmentation, but few data exist on the specific impact of ultraviolet radiation (UVR) on melanocytes in skin of different constitutive pigmentation. Objectives To analyse UVR‐induced DNA damage within melanocytes in different skin‐colour types. Methods Skin samples were objectively classified into light, intermediate, tan, brown and dark skin according to their individual typology angle (°ITA), based on colorimetric parameters. Samples were exposed to increasing doses of solar simulated radiation. Detection of DNA damage specifically in melanocytes was achieved by cyclobutane thymine dimer (CPD)–tyrosinase‐related protein 1 double staining. Results For light, intermediate and tan skin, accumulation of CPDs in melanocytes was detected at the lowest dose, with a steep increase with dose. At estimated erythemally equivalent doses, around 80–100% of melanocytes were positive for CPDs in tan, intermediate and light skin types. In contrast, in dark and brown skin types, CPDs were found in only approximately 15% of melanocytes at the highest dose. Conclusions This work demonstrates that melanocytes from constitutively highly pigmented skin types are less impacted in terms of UVR‐induced DNA damage than those from lighter skin types, even those that are moderately pigmented.     

Topical all-trans retinoic acid augments ultraviolet radiation-induced increases in activated melanocyte numbers in mice

We have previously shown that daily application of 0.05% retinoic acid to the backs of lightly pigmented, hairless HRA:Skh-2 mice increases melanogenesis resulting from exposure to solar-simulated ultraviolet radiation. In this study we show that as early as 1 wk following commencement of treatment, there is a 2- fold increase in the number of epidermal 3,4-dihydroxyphenylalanine positive melanocytes in retinoic acid and ultraviolet radiation treated HRA:Skh-2 mice compared with mice that received ultraviolet radiation only. This increased to a 2.9-fold difference by 6 wk. Retinoic acid also augmented ultraviolet radiation-stimulated melanogenesis, with a 4-fold increase being observed after only 2 wk. These findings were also seen in C57BL mice. Ultraviolet radiation and retinoic acid needed to be applied to the same skin site for the augmentation in melanocyte activation to occur. Ultraviolet B rather than ultraviolet A was mainly responsible for melanogenesis and the retinoic acid primarily increased ultraviolet B-induced melanogenesis. Furthermore, retinoic acid on it's own, in the absence of ultraviolet radiation caused a small but statistically significant increase in 3,4-dihydroxyphenylalanine positive melanocyte numbers and melanogenesis. Thus topical retinoic acid is a potent modulator of melanocyte activation. Alone it is able to increase the number of activated epidermal melanocytes and make melanocytes more sensitive to activation by ultraviolet B.     

The color of skin: white diseases of the skin,nails, and mucosa

White diseases are a heterogenous group characterized by hypopigmentation or depigmentation. Skin and eye color are determined by the number and size of melanosomes present. Melanin is produced by melanosomes in the melanocytes present within the epidermis of the skin, uvea, and retinal pigmented epithelium (RPE). Conditions altering the number of melanocytes or concentration of melanin result in a lack of pigmentation, appearing as “white diseases” ranging from the well-known albinism and vitiligo to more esoteric white hand syndrome and Degos disease.     

Disorders of Pigmentation

Skin color is highly individual and the variations are controlled by numerous genes. The different skin colors result from the size and number of melanosomes and do not mirror the amount of melanocytes. Disorders of pigmentation can result from migration abnormalities of melanocytes from the neural crest to the skin during embryogenesis. In addition, impairment of melanosome transfer to the surrounding keratinocytes, an alteration in melanin synthesis and a defective degradation or removal of melanin may lead to abnormal skin pigmentation. Immunologic or toxic mediated destructions of melanocytes can end in pigmentation disorders. Disorders of pigmentation are classified in hypo‐ or hyperpigmentation which can occur as a genetic or acquired disease. They can manifest locally or diffuse. Congenital hypopigmentation can be restricted to the skin as in piebaldism or they represent a systemic disease as in Menkes disease or phenylketonuria. Localized hypo‐ or hyperpigmentation in children may serve as markers for systemic diseases. Ash‐leaf hypopigmentation are characteristic for tuberous sclerosis and more than 5 café‐au‐lait spots suggest neurofibromatosis 1 (von Recklinghausen disease). The most common autoimmune‐induced depigmentation is vitiligo. Generalized hyperpigmentation only rarely reflects a primary genetic disorder but is most often from acquired diseases as in Addison disease, secondary hemochromatosis or primary biliary cirrhosis. Treatment of pigmentation disorders are based on a diagnosis which sometimes allow a specific intervention. Cosmetically acceptable results are difficult to obtain.     

Different effects of topically applied 5-fluorouracil on hairy and hairless mice

The effects of topically applied 5-fluorouracil (5-FU) ointment on hairy and hairless mice have been studied. Five per cent 5-FU ointment was applied once daily to the dorsal surface of the mice, totalling one, two or seven applications. No changes have occurred in all the hairy mice, while remarkable changes such as erosion or ulcer, followed by skin hypertrophy and pigmentation have occurred after the applications of the drug to hairless mice. In the altered hairless mice, acanthosis, the appearance of active (DOPA positive) melanocytes in the epidermis and dermal cyst wall, and moderate increase in the dermal melanocytes were seen in the marked pigmentation area. Pseudocarcinomatous hyperplasia and a high increase in the dermal melanocytes were seen in the hypertrophic skin. It is suggested that different from the hairy mice skin the hairless mice skin shows abnormal reaction to 5-FU (erosion or ulcer), resulting in epidermal hyperplasia and pigmentation.     

Topical W-7 inhibits ultraviolet radiation-induced melanogenesis in Skh:HR2 pigmented hairless mice

We studied the effect of N-(6-aminohexyl)-5-chloro-1-napthalenesulfonamide (W-7) on ultraviolet radiation (UVR)-induced melanogenesis (tanning) in Skh:HR2 pigmented hairless mice. Topically pretreated mice were exposed to subminimal edematogenic as well as edematogenic UVR doses to establish whether W-7-UVR-induced edema prophylaxis allows increased melanogenesis while preventing edema. Ultraviolet light-irradiated vehicle control animals developed visible tans; however, both W-7-treated groups failed to tan. Topical W-7 before UVR exposure inhibited UVR induction of dopa oxidase activity in melanocytes by 49% (P=0.029) and inhibited UVR-induced deposition of melanin in the epidermis by 88% (P=0.006). Topical W-7 blocked 23% of the UVR but this blockage could not account for the inhibition of dopa oxidase and melanization. We conclude that, in addition to preventing edema, W-7 inhibits UVR-induced melanogenesis, possibly by affecting Ca²⁺-calmodulin and/or protein kinase C-dependent processes.     

The hair follicle melanocytes in vitiligo in relation to disease duration

Background and aims  Vitiligo is an acquired pigmentary disorder of skin and hair. Active melanocytes in hair follicles can be detected by DOPA and immunohistochemical staining, while amelanotic melanocytes can only be detected by the latter. None of the studies on hair melanocytes in vitiligo discussed the effect of disease duration on these melanocytes.
Here, we study the presence of melanotic and amelanotic melanocytes in vitiligo hair follicles and statistically correlating their presence with the disease duration.
Methods  This study was conducted on 30 patients with vitiligo and 10 normal volunteers. Three biopsies were taken from each patient: two from black and white hairs from vitiliginous areas and the third from apparently normal skin of the same patients. Sections were stained by DOPA reaction and NKI/beteb then examined for the presence of melanocytes. The presence of melanocytes and the disease duration were correlated statistically using the t -test.
Results  Active melanocytes were detected in black hairs of 6.7% of vitiligo patients and in 100% of apparently normal skin of the same patients and controls. On examining black hairs of the 28 vitiligo patients with negative DOPA reaction, 19 of them (67.9%) showed positive NKI/beteb stain. Disease duration was inversely correlated with the melanocytes' presence within hair follicles. Melanocytes were absent from 100% of white hairs.
Conclusions  The melanotic melanocytes were the first target of the disease process followed by the amelanotic melanocytes. Since the disappearance of the latter was inversely correlated with the disease duration, early treatment in vitiligo is advised.

Conflicts of interest

None declared.     

Molecular and cellular basis of depigmentation in vitiligo patients

Vitiligo is a chronic skin disease characterized by the appearance of zones of depigmentation. It is mostly described as an autoimmune disease in which the immune system destroys the melanocytes. Consistent with this origin, genetic studies have implicated genes encoding proteins mediating the immune response targeting melanocytes in the aetiology of this disease, together with proteins specific to these cells. However, the destruction of melanocytes by the immune system is neither global nor complete, because the patients do not display total depigmentation. The etiopathology of vitiligo is clearly complex and cannot be simply reduced to an autoimmune reaction directed against pigmented cells. Intrinsic changes have been observed in the melanocytes, keratinocytes and dermal cells of vitiligo patients. Identification of the molecular and cellular changes occurring in normally pigmented skin in vitiligo patients, and an understanding of these changes, is essential to improve the definition of trigger events for this disease, with a view to developing treatments with long‐term efficacy. This review focuses on the early events identified to date in the non‐lesional regions of the skin in vitiligo patients and discusses the process of repigmentation from melanocyte stem cells.     

Development of melanocye-keratinocyte co-culture model for controls and vitiligo to assess regulators of pigmentation and melanocytes

Background: There is a need to develop an in vitro skin models which can be used as alternative system for research and testing pharmacological products in place of laboratory animals. Therefore to study the biology and pathophysiology of pigmentation and vitiligo, reliable in vitro skin pigmentation models are required. Aim: In this study, we used primary cultured melanocytes and keratinocytes to prepare the skin co-culture model in control and vitiligo patients. Methods: The skin grafts were taken from control and patients of vitiligo. In vitro co-culture was prepared after culturing primary melanocytes and keratinocytes. Co- cultures were treated with melanogenic stimulators and inhibitors and after that tyrosinase assay, MTT assay and melanin content assay were performed. Results: Melanocytes and keratinocytes were successfully cultured from control and vitiligo patients and after that co-culture models were prepared. After treatment of co-culture model with melanogenic stimulator we found that tyrosinase activity, cell proliferation and melanin content increased whereas after treatment with melanogenic inhibitor, tyrosinase activity, cell proliferation and melanin content decreased. We also found some differences in the control co-culture model and vitiligo co-culture model. Conclusion: We successfully constructed in vitro co-culture pigmentation model for control and vitiligo patients using primary cultured melanocytes and keratinocytes. The use of primary melanocytes and keratinocytes is more appropriate over the use of transformed cells. The only limitation of these models is that these can be used for screening small numbers of compounds.     

Role of hair follicles in the repigmentation of vitiligo.

Vitiligo is a common pigment disease that is difficult to treat. The mechanism of repigmentation is not known. We combined Dopa-Toluidine Blue complex stain, hair follicle split-Dopa stain, and hair follicle split-scanning electron microscope (SEM) to observe the changes of melanocytes in 23 normal, 24 vitiliginous, and 36 repigmented skin specimens. We found that only active (Dopa-positive) melanocytes existed in the epidermis of normal skin. There were some inactive (Dopa-negative) melanocytes in the outer root sheaths of normal hair follicles, which form the melanocyte reservoir in human skin. In the patients with vitiligo the active melanocytes in the epidermis were totally missing, whereas the inactive melanocytes in the outer root sheaths of hair follicles were not affected. Treatment stimulated the inactive melanocytes in the middle and/or lower parts of the outer root sheaths of hair follicles to divide, proliferate, and migrate upward along the surface of the outer root sheath to the nearby epidermis, where the melanocytes continued to migrate radially to form the pigmented island visible clinically in repigmented vitiligo lesions. During the migration to the epidermis, the melanocytes matured gradually from an inactive phase to an active condition. In conclusion, the existence of these inactive melanocytes provided the melanocyte sources for repigmentation of vitiligo.     

Possible mechanisms of hypopigmentation in lichen sclerosus

Lichen sclerosus (LS) shares with vitiligo a milky-white appearance. By biopsy, pathognomonic dermal sclerosis readily distinguishes LS from vitiligo and other causes of leukoderma. To determine what the mechanism of hypopigmentation is in LS, we examined samples from LS cases for alterations in melanin content (Fontana-Masson stain) and melanocyte number (HMB-45 [PMEL-17/gp100], Mel-5 [TRP-1], Mart-1 [Melan A]) and compared these findings with those in controls of normal skin, acute scars, vitiligo, and lichen planus (LP; a common inflammatory cause of hyperpigmentation). The degree and extent of melanization found in LS overlapped with that in acute scars showing predominantly hypomelanized keratinocytes, with that in LP containing regions with numerous melanophages, and with that in vitiligo exhibiting focal regions of keratinocytes devoid of melanin pigment. By hematoxylin-eosin staining and immunocytochemistry for Mel-5 and Mart-1, LS had a lower mean count of melanocytes than acute scars, LP, and normal skin per 200 basal keratinocytes. In addition, a few LS cases had a significant loss of melanocytes comparable to that of vitiligo. Surprisingly, Mart-1 identified rare melanocytes in 67% of vitiligo cases and a significantly larger pool of melanocytes in LS and controls other than those labeled by Mel-5. Furthermore, LP and evolving lesions of LS contained the highest Mart-1 counts. HMB-45-immunoreactive melanocytes were found in the majority of acute scars and in LP and late-stage LS lesions at significantly lower levels than Mel-5- and Mart-1- labeled melanocytes, but they were not found in vitiligo or normal skin. We propose that several mechanisms may play a role in the production of leucoderma in LS: 1) decreased melanin production; 2) block in transfer of melanosomes to keratinocytes; and 3) melanocyte loss. The latter finding may be the pathogenic connection (lichenoid dermatitis of LS triggering an autoimmune reaction to melanocytes) that underlies the documented association of LS with vitiligo.     

Patterns of repigmentation in two cases of hypopigmented type of vitiligo

It has been observed that depigmentation in vitiligo passes through two stages; patches of light brown hypopigmentation which gradually changes into milky white patches. In this work, we studied two cases of hypopigmented vitiligo regarding the melanocytes and keratinocytes' changes before and after 7 months of psoralen plus ultraviolet A (PUVA) therapy. Skin biopsies were taken from the vitiliginous lesions before and after 7 months of PUVA therapy and were examined using haematoxylin and eosin and Masson Fontana stains, l -3,4-dihydroxyphenylalanine reaction, immuno-histochemical stains and ultrastructural examination. In the pretreated patients, the melanocytes present were inactive and degenerative changes were detected in both melanocytes and keratinocytes. After PUVA therapy, obvious histopathological improvement was detected. Clinically, the initial response to PUVA therapy was increased hypopigmentation indicating that degenerated cells in the vitiliginous patches might have continued the process of degeneration and did not recover. Meanwhile, the perifollicular and marginal pigmentation suggested that pigmentation occurred from those areas and not from activation of already degenerated melanocytes.     

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.     

Autologous melanocyte–keratinocyte suspension in the treatment of vitiligo

Background In stable vitiligo, several techniques of autologous transplantation of melanocytes are used. Autologous melanocyte transplantation of non‐cultured melanocytes is one of those techniques with variable reported outcomes. Objective The objective of this study was to evaluate the response to autologous melanocyte–keratinocytes suspension transplantation in cases of stable vitiligo. Methods A total of 25 cases of vitiligo were treated by autologous melanocyte–keratinocytes suspension transplantation. After 6–17 months, patients’ response was evaluated according to the extent of pigmentation (excellent 90–100%, good 50–89%, fair 20–49% and poor response <20%). Results Of the 25 patients treated, 22 continued the follow‐up period. Five (23%) patients showed excellent response, 7 (32%) good, 6 (27%) fair and 4(18%) showed poor response. Conclusion Unlike transplantation of cultured melanocytes, which requires experience in culture technique, autologous melanocyte–keratinocytes suspension transplantation is an easy economic technique, which may be used in resistant areas of stable vitiligo.     

4‐(4‐Hydroxyphenyl)‐2‐butanol (rhododendrol)‐induced melanocyte cytotoxicity is enhanced by UVB exposure through generation of oxidative stress

4‐(4‐Hydroxyphenyl)‐2‐butanol (rhododendrol, RD), a skin‐whitening agent, was reported to cause skin depigmentation in some users, which is attributed to its cytotoxicity to melanocyte. It was reported that cytotoxicity to melanocyte is possibly mediated by oxidative stress in a tyrosinase activity‐dependent manner. We examined the effect of UV radiation (UVR) on RD‐induced melanocyte cytotoxicity as an additional aggravating factor. UVR enhanced RD‐induced cytotoxicity in normal human epidermal melanocytes (NHEMs) via the induction of endoplasmic reticulum (ER) stress. Increased generation of intracellular reactive oxygen species (ROS) was detected. Pretreatment with N‐acetyl cysteine (NAC), antioxidant and precursor of glutathione significantly attenuated ER stress‐induced cytotoxicity in NHEMs treated with RD and UVR. Increase in cysteinyl‐RD‐catechol and RD‐pheomelanin in NHEMs treated with RD and UVR suggested that, after UVR excitation, RD or RD metabolites are potent ROS‐generating substances and that the tendency to produce RD‐pheomelanin during melanogenesis amplifies ROS generation in melanocytes. Our results help to elucidate the development mechanisms of RD‐induced leukoderma and provide information for innovation of safe skin‐whitening compounds.     

Differential analysis of experimental hypermelanosis induced by UVB,PUVA, and allergic contact dermatitis using a brownish guinea pig model

Summary In moderately colored guinea-pig skin, UVB, PUVA, and allergic contact dermatitis were shown to induce hyperpigmentation that resembled the pigmentary changes observed in mongoloid human skin. Using this model, we examined the effects of chemical agents, including tyrosinase inhibitors and sunscreen agents, on the color changes induced by UV irradiation. The daily exposure of brownish guinea-pig skin to UVB irradiation at a variety of energies for 3 successive days induced clearly visible black pigmentation on the irradiated rectangular areas of the flank within a few days of irradiation, the maximum being reached about 1 week after irradiation, i.e., similar to the changes that occur in pigmented human skin. Split epidermal sheets prepared from untreated pigmented guinea pigs exhibited 200–400 melanocytes/mm²; 1 week after UV irradiation, the applied areas show an increased number of strongly dopa-positive melanocytes with stout dendrites (800–1,000 cells/mm²). UVA irradiation following an intraperitoneal injection of 8-methoxypsoralen (8-MOP) also produced black pigmentation 1 week after irradiation, and this was paralleled by a marked increase in the number of dopa-positive melanocytes in dopa-reacted split epidermal sheets. Allergic contact dermatitis produced by the application of 1-phenylazo-2-naphtol induced hyperpigmentation after an interval of about 14 days in 10 of the 21 allergy-acquiring animals examined. This induced pigmentation was accompanied by an increase in the number of dopa-positive melanocytes as compared to the number seen in controls. In contrast, allergic contact dermatitis produced by the application of dinitrochlorobenzene failed to induce such a high ratio of postpigmentation, with only 3 of the 21 allergy-acquiring animals showing hyperpigmentation and 5 showing depigmentation; in the latter, there was a slight decrease in the number of dopa-positive melanocytes. To study the preventive effect of tyrosine inhibitors on UVB-induced pigmentation, daily topical applications of these compounds were performed after three daily UVB irradiations. Treatment with 10% hydroquinone for 10 days interrupted UVB-induced pigmentation and resulted in a marked reduction in the number of epidermal melanocytes as compared to the number found in UVB-irradiated, untreated control skin.     

Delayed induction of pigmented spots on UVB-irradiated hairless mice

Human skin exposed to solar radiation for a long time subsequently develops pigmented spots, which are named solar lentigines. Since no animal model of this process is currently available, we attempted to induce similar spots in pigmented hairless mice. The mice were irradiated at 38 or 94 mJ/cm(2) three times/week for various periods of time (1-8 weeks) under an ultraviolet light source (Toshiba FL-SE; UVB). Skin pigmentation of irradiated mice was visually observed and skin color was determined with a colorimeter for 78 weeks. Uniform pigmentation was induced, but persisted only during exposure, disappearing completely within 2 weeks after cessation of exposure. At about 28 weeks after the first exposure, pigmented spots suddenly began to appear. These pigmented spots were less than 2 mm in diameter and light brown in color. The length of the latent period until appearance and the extent of development of these spots were dependent on the exposure period. Histological examination revealed increased numbers of active melanocytes and melanin granules in the affected epidermis. These pigmented spots closely resemble solar lentigines in humans, and the mice should be useful as an animal model of solar lentigines.     

Accelerating bleaching in vitiligo: balancing benefits versus risks

While the goal of available treatment in vitiligo is to regain pigmentation, some patients affected by extensive and treatment‐resistant vitiligo, with a major social and emotional impact, may benefit from depigmentation therapy. However, results from such therapy may not always be satisfactory. So to achieve better, faster and complete bleaching, Webb et al. propose a synergistic approach that combines topical application of bleaching phenols which targets melanocytes and initiate local inflammation with immune adjuvants so as to obtain an enhanced immune response against remaining melanocytes. This strategy could be reliable, but should be evaluated cautiously in future studies, in terms of potential side effects and induction of undesired autoimmunity.     

Successful Treatment of Vitiligo on the Scalp of a 9‐Year‐Old Girl Using Autologous Cultured Pure Melanocyte Transplantation

Leukotrichia frequently accompanies vitiligo on hairy areas such as the scalp. Treatment with conventional medical therapy is usually unsuccessful because of deficiencies in the melanocyte reservoir. We describe transplantation of autologous cultured pure melanocytes for scalp vitiligo with leukotrichia in a 9‐year‐old girl, resulting in almost complete and stable repigmentation of skin and hair.