Melanin content of cultured human melanocytes and UV-induced cytotoxicity

Cultured melanocytes originating from persons with different skin phototypes were utilized for measurement of endonuclease sensitive sites induced by UVB and the determination of cell survival after UVA or UVB irradiation. During culture, the melanocytes largely maintained their phenotypic characteristics according to their original skin phototype. Total melanin concentrations were 4.9 times higher in the darker skin phototype (IV-VI) melanocytes when compared to the cells from lighter skin phototypes (I-III). Also phaeomelanin contents were higher (2.5 times) in the skin phototype (IV-VI) melanocytes which implies that the cells from light skin types contain less melanin, but a relatively high proportion of phaeomelanin. After UVB irradiation a stronger induction of endonuclease sensitive sites was found for melanocytes with a lower level of total melanin and a high content of pheomelanin. By measuring the clone forming ability in different melanocyte cultures after UVB irradiation, significant better survival was found in case of the cells with the higher melanin content. Despite the large variations in melanin content, no significant difference in survival after UVA irradiation could be demonstrated in this way. Our results suggest a protective effect of melanin for UVB and indicate the importance of the measurements of melanin content and composition when different parameters of UV-induced damage are studied in melanin producing cells.     

Role of UV in cutaneous melanoma

Malignant melanoma arises from epidermal melanocytes, the cells responsible for the production of the skin pigment melanin. The photoprotective role of melanin, which is transferred to neighboring keratinocytes, in UV-induced skin carcinogenesis, specifically in nonmelanoma skin cancers, has been well documented. Although melanocyte-resident melanin is expected to offer similar protection to melanocytes from UV-induced damage, UV radiation has long been suspected to have an etiologic role in cutaneous melanoma. However, nearly three decades of efforts using a variety of in vitro and in vivo models of human skin and mouse genetic models have produced conflicting data. Epidemiologic studies have also failed to establish a definitive association between UV exposure and risk of melanoma. In this review, we evaluate the dual role of the melanin pigment as a photoprotector as well as a photosensitizer and examine the evidence for association between melanin levels (constitutive and induced) and melanoma risk. We also discuss possible reasons for the lack of signature UV mutations in melanoma oncogenes known to date and potential alternative mechanisms to explain the role of UV in melanomagenesis.     

Photobiology of ocular melanocytes and melanoma

There are two different types of ocular melanocytes and melanomas. Conjunctival melanocytes are located on the surface of the eye and are exposed to visible light and UV radiation. Recently, epidemiological studies demonstrated that sunlight plays a definite role in the occurrence of conjunctival melanoma, as it does in cutaneous melanoma. Uveal melanocytes consist of the iridal melanocytes, which are exposed to visible light and UV radiation; and the ciliary body melanocytes and choroidal melanocytes, which are not exposed to light radiation. Epidemiological studies demonstrated that sunlight may play a role in the occurrence of iridal melanoma, but may not be a major factor in the etiology of ciliary body and choroidal melanomas. Uveal melanocytes differ from epidermal melanocytes in that epidermal melanocytes respond to UV radiation and skin color becomes darker after exposure to sunlight; but uveal melanocytes do not respond to UV radiation and the iris color remains stable after exposure to sunlight. Recently, in vitro studies indicate that this phenomenon is determined both by cellular factors and environmental factors.     

DNA Damage, Repair and Photoadaptation in a Xiphophorus Fish Hybrid

Exposure to sunlight is responsible for most cutaneous malignant melanomas in the human population. It is very likely that DNA damage is an initial event in melanomagenesis, however, the role played by this damage is an open question. To this end, we used a hemipigmented F₁ hybrid of the fish genus Xiphophorus and HPLC tandem mass spectrometry to examine the effects of melanin on the induction and repair of the predominant UV-induced photoproducts formed in skin cell DNA. We found that heavily pigmented skin cells had about half the damage of nonpigmented cells and the relative induction of the major photoproducts was independent of the degree of pigmentation. The efficiency of photoenzymatic repair was the same in nonpigmented and pigmented areas of the fish. We found no evidence of residual damage at 10 days after the last exposure. Most striking was that repeated exposure to multiple doses of UVB caused a very significant photoadaptive response. Rather than an accumulation of damage after five doses of UVB we saw a significant reduction in the amount of damage induced after the final dose compared with the initial dose. The relevance of these observations is discussed in the context of melanoma susceptibility and UVB thresholds.     

THE UV ACTION SPECTRA FOR THE CLONE-FORMING ABILITY OF CULTURED HUMAN MELANOCYTES AND KERATINOCYTES

Abstract Melanocytes (skin type 2) and keratinocytes were irradiated with UV light of 254, 297, 302, 312 and 365 nm and the survival was measured. Clone-forming ability was chosen as the parameter for cell survival. Melanocytes were found to be less sensitive to UV light than keratinocytes (a difference of a factor 1.22-1.92 for the UV-C and UV-R wavelengths (254, 297, 301 and 312 nm) and a factor 6.71 for the UV-A wavelength (365 nm). Because melanin does not appear to protect against the induction of pyrimidine dimers the difference between melanocytes and keratinocytes in the UV-C and UV-B region could not be explained by the presence of melanin in the melanocytes. The relatively small difference can be explained by the longer cell cycle of melanocytes, which provides more time for the melanocytes to repair UV damage. In the UV-A region the difference between melanocytes and keratinocytes was much larger, suggesting that besides the longer cell cycle some additional factors must be involved in protection against UV-A light.     

The melanocortin 1 receptor and the UV response of human melanocytes--a shift in paradigm

Cutaneous pigmentation is the major photoprotective mechanism against the carcinogenic and aging effects of UV. Epidermal melanocytes synthesize the pigment melanin, in the form of eumelanin or pheomelanin. Synthesis of the photoprotective eumelanin by human melanocytes is regulated mainly by the melanocortins alpha-melanocortin (alpha-MSH) and adrenocorticotropic hormone (ACTH), which bind the melanocortin 1 receptor (MC1R) and activate the cAMP pathway that is required for UV-induced tanning. Melanocortins stimulate proliferation and melanogenesis and inhibit UV-induced apoptosis of human melanocytes. Importantly, melanocortins reduce the generation of hydrogen peroxide and enhance repair of DNA photoproducts, independently of pigmentation. MC1R is a major contributor to the diversity of human pigmentation and a melanoma susceptibility gene. Certain allelic variants of this gene, namely R151C, R160W and D294H, are strongly associated with red hair phenotype and increased melanoma susceptibility. Natural expression of two of these variants sensitizes melanocytes to the cytotoxic effect of UV, and increases the burden of DNA damage and oxidative stress. We are designing potent melanocortin analogs that mimic the effects of alpha-MSH as a strategy to prevent skin cancer, particularly in individuals who express MC1R genotypes that reduce but do not abolish MC1R function, or mutations in other melanoma susceptibility genes, such as p16.     

Melanin offers protection against induction of cyclobutane pyrimidine dimers and 6-4 photoproducts by UVB in cultured human melanocytes

The goal of this investigation was to correlate the melanin content in human pigmentary cells with the generation of UVB-induced photoproducts and to examine the relationship between the melanin content and the removal of the photoproducts. Cultured melanocytes from light-skinned individuals synthesized less melanin and produced more cyclobutane pyrimidine dimers and 6-4 photoproducts upon UVB exposure than did melanocytes from black skin. Tyrosine-stimulated melanogenesis provided protection against DNA damage in both cell types. In another set of pigmented cell lines a ratio between eumelanin and pheomelanin was determined. The assessment of association between DNA damage induction and the quantity and quality of melanin revealed that eumelanin concentration correlated better with DNA protection than pheomelanin. Skin type-I and skin type-VI melanocytes, congenital nevus (CN)-derived cells and skin type-II melanocytes from a multiple-melanoma patient were grown in media with low or high L-tyrosine concentration. The cells were irradiated with 200 J/m2 UVB, and the levels of the photoproducts were determined immediately and after 6 and 24 h. Once again the induction of the photoproducts was mitigated by increased melanogenesis, and it was inversely correlated with the skin type. No significant differences were found for the removal of photoproducts in the cultures of skin types I and VI and CN cells. No indications of a delay in the removal of photoproducts in the melanocytes from the multiple-melanoma patient were found either.     

MC1R,Eumelanin and Pheomelanin: Their Role in Determining the Susceptibility to Skin Cancer

Skin pigmentation is due to the accumulation of two types of melanin granules in the keratinocytes. Besides being the most potent blocker of ultraviolet radiation, the role of melanin in photoprotection is complex. This is because one type of melanin called eumelanin is UV absorbent, whereas the other, pheomelanin, is photounstable and may even promote carcinogenesis. Skin hyperpigmentation may be caused by stress or exposure to sunlight, which stimulates the release of α‐melanocyte stimulating hormone (α‐MSH) from damaged keratinocytes. Melanocortin 1 receptor (MC1R) is a key signaling molecule on melanocytes that responds to α‐MSH by inducing expression of enzymes responsible for eumelanin synthesis. Persons with red hair have mutations in the MC1R causing its inactivation; this leads to a paucity of eumelanin production and makes red‐heads more susceptible to skin cancer. Apart from its effects on melanin production, the α‐MSH/MC1R signaling is also a potent anti‐inflammatory pathway and has been shown to promote antimelanoma immunity. This review will focus on the role of MC1R in terms of its regulation of melanogenesis and influence on the immune system with respect to skin cancer susceptibility.     

COMPARATIVE STUDIES ON THE CORRELATION BETWEEN PYRIMIDINE DIMER FORMATION and TYROSINASE ACTIVITY IN CLOUDMAN S91 MELANOMA CELLS AFTER ULTRAVIOLET-IRRADIATION

We compared the induction of pyrimidine dimer densities after UV-irradiation in mouse melanoma cells before and after treatment with cholera toxin. Treatment with cholera toxin stimulated tyrosinase activity up to 50-fold, leading to a marked, visually apparent increase in cellular melanin concentrations. Irradiation of treated and untreated cells was therefore designed to establish whether intracellular melanin protected cells from UV-induced DNA damage. In experiments described here, we determined cytosine-thymine (C-T) as well as thymine-thymine dimer levels (T-T) by high pressure liquid chromatography in cholera toxin-treated and untreated Cloudman S91 mouse melanoma cells after irradiation with UVC (less than 290 nm) and UVB light (290-320 nm). Surprisingly, induction of melanization had no effect on the formation of pyrimidine dimers by UVC or UVB irradiation. These results indicate that de novo melanin pigmentation induced via the c-AMP pathway is not involved in protection against UV-induced thymine-containing pyrimidine dimers. In separate experiments, irradiation of toxin-treated and untreated mouse melanoma cells with UVC or UVB light produced a 20-30% lower dimer density compared to irradiated human skin fibroblasts. This finding suggests that melanin has some protection properties against UV-induced pyrimidine dimers, although the exact defense mechanism seems highly complex.     

Epidemiological Support for an Hypothesis for Melanoma Induction Indicating a Role for UVA Radiation

An hypothesis for melanoma induction is presented: UV radiation absorbed by melanin in melanocytes generates products that may activate the carcinogenic process. Products formed by UV absorption in the upper layers of the epidermis cannot diffuse down as far as to the melanocytes. Thus, melanin in the upper layer of the skin may be protective, while that in melanocytes may be photocarcinogenic. Observations that support this hypothesis include: (1) Africans with dark skin have a reduced risk of getting all types of skin cancer as compared with Caucasians, but the ratio of their incidence rates of cutaneous malignant melanoma to that of squamous cell carcinoma is larger than the corresponding ratio for Caucasians. (2) Albino Africans, as compared with normally pigmented Africans, seem to have a relatively small risk of getting cutaneous malignant melanomas compared to nonmelanomas. This is probably also true for albino and normally pigmented Caucasians. (3) Among sun-sensitive, poorly tanning persons, frequent UV exposures are associated with increased risk of melanoma, whereas among sun-resistant, well-tanning persons, increased frequency of exposure is associated with decreased melanoma risk. (4) It is likely that UVA, being absorbed by melanin, might have a melanoma-inducing effect. This is in agreement with some epidemiological investigations which indicate that sun-screen lotions may not protect sufficiently against melanoma induction. The relative latitude gradient for UVA is much smaller than that for UVB. The same is true for the relative latitude gradient of cutaneous malignant melanoma as compared with squamous cell carcinoma and basal cell carcinoma. Under the assumption that the average slopes of the curves relating incidence rates with fluences of carcinogenic UV radiation are similar for melanomas and nonmelanomas, these facts are in agreement with the assumption that UVA plays a significant role in the induction of melanomas in humans. This is in agreement with the experimental results with Xiphophorus.     

Radiation sources providing increased UVA/UVB ratios induce photoprotection dependent on the UVA dose in hairless mice

In studies involving mice in which doses of UVA (320-400 nm) and UVB (290-320 nm) radiation were administered alone or combined sequentially, we observed a protective effect of UVA against UVB-induced erythema/edema and systemic suppression of contact hypersensitivity. The UVA immunoprotection was mediated by the induction of the stress enzyme heme oxygenase-1 (HO-1) in the skin, protection of the cutaneous Th1 cytokines interferon-gamma (IFN-gamma) and IL-12 and inhibition of the UVB-induced expression of the Th2 cytokine IL-10. In this study, we seek evidence for an immunological waveband interaction when UVA and UVB are administered concurrently to hairless mice as occurs during sunlight exposure in humans. A series of spectra providing varying ratios of UVA/UVB were developed, with the UVA ratio increased to approximately 3.5 times the UVA component in solar simulated UV (SSUV). We report that progressively increasing the UVA component of the radiation while maintaining a constant UVB dose resulted in a reduction of both the erythema/edema reaction and the degree of systemic immunosuppression, as measured as contact hypersensitivity. The UVA-enhanced immunoprotection was abrogated in mice treated with a specific HO enzyme inhibitor. UVA-enhanced radiation also upregulated the expression of cutaneous IFN-gamma and IL-12 and inhibited expression of both IL-6 and IL-10, compared with the activity of SSUV. The results were consistent with the previously characterized mechanisms of photoprotection by the UVA waveband alone and suggest that the UVA component of solar UV may have beneficial properties for humans.     

Skin Erythema,Pigmentation and Hydration Kinetics after Ultraviolet Radiation‐induced Photodamage in Southern Chinese Women

Although there have been some studies about changes of skin erythema and pigmentation following ultraviolet radiation in other races, the relevant data in Chinese have never been achieved. Thus, we evaluated the long‐time course of skin erythema, pigmentation and hydration changes after different doses of solar‐simulated ultraviolet (SSUV) irradiation in 26 Chinese women for 168 days. The erythema index increased abruptly and peaked during 3 days of SSUV exposure, then slowly returned to the baseline level starting at day 7 and completely recovered during 168‐day course of this study only in one minimal erythema doses (MED) SSUV irradiation. The melanin index started to slowly increase at day 3 of SSUV exposure, peaking at day 14 and gradually returned to the baseline level thereafter, but did not return to the baseline level during 168‐day course in all doses. Skin hydration slowly declined at day 3 of exposure, hitting the lowest point at day 7, then slowly recovered starting at day 14 and completely returned to the baseline level at day 28 only in 1.5MED. These results will serve as baseline data on Chinese skin and provide useful references for the treatment of serious skin photodamage in Chinese.     

Distinct Role of Sesn2 in Response to UVB‐Induced DNA Damage and UVA‐Induced Oxidative Stress in Melanocytes

Ultraviolet (UV) radiation, including both UVB and UVA irradiation, is the major risk factor for causing skin cancer including melanoma. Recently, we have shown that Sesn2, a member of the evolutionarily conserved stress‐inducible protein family Sestrins (Sesn), is upregulated in human melanomas as compared to melanocytes in normal human skin, suggesting an oncogenic role of Sesn2. However, the role of Sesn2 in UVB and UVA response is unknown. Here, we demonstrated that both UVB and UVA induce Sesn2 upregulation in melanocytes and melanoma cells. UVB induces Sesn2 expression through the p53 and AKT3 pathways. Sesn2 negatively regulates UVB‐induced DNA damage repair. In comparison, UVA induces Sesn2 upregulation through mitochondria but not Nrf2. Sesn2 ablation increased UVA‐induced Nrf2 induction and inhibits UVA‐induced ROS production, indicating that Sesn2 acts as an upstream regulator of Nrf2. These findings suggest previously unrecognized mechanisms in melanocyte response to UVB and UVA irradiation and potentially in melanoma formation.     

Photochemistry and photobiology of melanogenic metabolites: formation of free radicals

Normal melanocytes produce specialized subcellular organelles called melanosomes within which the biochemical processes of melanogenesis occurs. During sunlight-induced melanogenesis, the melanocyte-specific enzyme tyrosinase catalyzes the oxidative polymerization of 3,4-dihydroxyphenyl-alanine (DOPA) to melanin. Nucleophilic addition of cysteine to tyrosinase-generated dopaquinone leads to the formation of cysteinyldopas, precursors of pheomelanin and excreted by-products of eumelanogenesis. Under conditions of low sulfhydryl content, dopaquinone undergoes a 1,4 intramolecular cycloaddition to yield, after further oxidation, 5,6-dihydroxyindoles and/or 5,6-dihydroxy-2-carboxyindoles. These indolic melanogenic intermediates and their O-methylated metabolites, like cysteinyldopas, are excreted by actively pigmenting as well as dormant melanocytes. Indeed, it has been determined that in humans, the serum and urine concentrations of these melanogenic metabolites increase dramatically following exposure to sunlight, UVA (315-400 nm), UVB (290-315 nm) exposure, as well as during PUVA therapy and in melanoma patients, and thus have proved to be excellent biochemical markers of normal and pathological melanocyte function. While controlled light exposure or PUVA therapy generally lead to 100-300% increases in 5-S-cysteinyldopa (5SCD) and 5-methoxy-6-hydroxyindole-2-carboxylic acid (6HMICA) serum levels (normal concentration about 4–16 nmol l^-1), the local concentrations in the skin and especially in the actively pigmenting melanocyte may be as high as 200 μM. Evidence is presented to document that a number of catecholic melanin precursors, including cysteinyldopas and dihydroxyindoles, are photochemically unstable in the presence of biologically relevant ultraviolet radiation (i.e. wavelengths ± 300 nm). Initial photochemical processes involve free radical production; continued photolysis yields polymeric photoproducts. Radicals produced during melanogenic metabolite photolysis have been identified by ESR spin trapping, laser flash photolysis and pulse radiolysis techniques and include hydrated electrons (e_aq), hydrogen atoms (H'), hydroxyl radicals (OH), semiquinones, aryl thiyl (ArS), and alanyl carbon-based radicals. In vitro investigations of the potential photobiological significance of these reactions have demonstrated photolysis of cysteinyldopas may lead to photoinitiated DNA binding and single strand break induction. The above mentioned radical species may also damage proteins and initiate lipid peroxidation. Definitive evidence for the occurrence of these phototoxic reactions in vivo is currently unavailable, however our in vitro studies suggest a possible role for melanogenic metabolite photolysis in acute and chronic solar responses of human skin.     

1-(2,4-dihydroxyphenyl)-3-(2,4-dimethoxy-3-methylphenyl)propane, a novel tyrosinase inhibitor with strong depigmenting effects

A series of diarylpropane compounds was isolated by screening a plant extract library for inhibitors of mushroom tyrosinase. The most potent compound, 1-(2,4-dihydroxyphenyl)-3-(2,4-dimethoxy-3-methylphenyl)propane (UP302: CAS# 869743-37-3), was found in the medicinal plant Dianella ensifolia. Synthetic and plant-derived versions of UP302 inhibited mushroom tyrosinase with similar potencies. UP302 inhibited mushroom tyrosinase with K(i)=0.3 microM, in a competitive and reversible fashion. UP302 was 22 times more potent than Kojic acid in inhibiting murine tyrosinase, with IC(50) values of 12 and 273 microM respectively. Experiments on mouse melanoma cells B16-F1 and on human primary melanocytes demonstrated that UP302 inhibits melanin formation with IC(50) values of 15 and 8 microM respectively. Long-term treatment of cultured melanocytes with up to 62 microM of UP302 revealed no detectable cytotoxicity. In a reconstructed skin model (MelanoDerm) topical application of 0.1% UP302 resulted in significant skin lightening and decrease of melanin production without effects on cell viability, melanocyte morphology or overall tissue histology. In conclusion, UP302 is a novel tyrosinase inhibitor that suppresses melanin production in both cultured melanocytes and reconstructed skin with high potency and without adverse side effects.     

The Human Melanocyte as a Particular Target for UVA Radiation and an Endpoint for Photoprotection Assessment

The induction of DNA breaks by UVA (320-400 nm) in the nucleus of normal human melanocytes in culture was investigated using single cell gel electrophoresis, also called the comet assay. Endogenous pigment and/or melanin-related molecules were found to enhance DNA breakage: comets were more intense in melanocytes than in fibroblasts, in cells with high melanin content or after stimulation of melanogenesis by supplying tyrosine in the culture medium. After UVA doses where strong comets were observed, neither cytotoxicity nor stimulation of tyrosinase activity were detected. However, the accumulation of p53 protein suggested that cells reacted to genotoxic stress under these experimental conditions. The same approach was used to compare two sunscreens with identical sun protection factors but different UVA protection factors. The results presented in this paper suggest that human melanocytes may be used as a target cell to evidence broadspectrum photoprotection. Moreover, these data appear to be helpful in getting a better understanding of the role of sunlight in the initiating steps of melanocyte transformation.     

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.     

UV irradiation affects melanocyte stimulatory activity and protein binding of piperine

Piperine, the major alkaloid of black pepper (Piper nigrum L.; Piperaceae), stimulates melanocyte proliferation and dendrite formation in vitro. This property renders it a potential treatment for the skin depigmentation disorder vitiligo. However, piperine does not stimulate melanin synthesis in vitro, and treatments based on this compound may therefore be more effective with concomitant exposure of the skin to ultraviolet (UV) radiation or sunlight. The present study investigated the effect of UVA and simulated solar radiation (SSR) on the chemical stability of piperine, its melanocyte stimulatory effects and its ability to bind protein and DNA. Chromatographic and spectroscopic analysis confirmed the anticipated photoisomerization of irradiated piperine and showed the absence of any hydrolysis to piperinic acid. Isomerization resulted in the loss of ability to stimulate proliferation of a mouse melanocyte cell line, and to bind to human serum albumin. There was no evidence of DNA binding by piperine either before or after irradiation, showing the absence of photoadduct formation by either piperine or its geometric isomers. This is unlike the situation with psoralens, which form DNA adducts when administered with UVA in treating skin diseases. The present study suggests that exposure to bright sunlight should be avoided both during active application of piperine to the skin and in the storage of piperine products. If UVA radiation is used with piperine in the treatment of vitiligo, application of the compound and irradiation should be staggered to minimize photoisomerization. This approach is shown to effectively induce pigmentation in a sparsely pigmented mouse strain.